Submitted by Claudinei Pracidelli (cpracide@ipen.br) on 2015-04-10T14:16:47Z No. of bitstreams: 0 / Made available in DSpace on 2015-04-10T14:16:47Z (GMT). No. of bitstreams: 0 / O desenvolvimento de nanosensores luminescentes, não tóxicos e biocompatíveis estão sendo utilizados para marcação e visualização de células em biologia celular e imunoensaios ultrassensíveis. Neste trabalho, marcadores biológicos à base de terras raras para a detecção do 17-β-estradiol (E2), em fluido biológico(plasma) foram estudados. Os complexos precursores à base de terras raras (Eu3+ e Tb3+) foram sintetizados e caracterizados. O complexo contendo tta- de Eu3+ e liganteauxiliar TOPO [Eu(tta)3(TOPO)2] registrou a maior eficiência de luminescência dentre todos os demais compostos estudados e foi escolhido como sonda luminescente para desenvolvimento das etapas seguintes deste trabalho. Este complexo foi incorporado às matrizes poliméricas, PMMA e PHB, em diferentes concentrações de dopagens pordois métodos distintos: nanoprecipitação crioscópica e pela técnica de emulsão-difusão. As nanopartículas poliméricas de PMMA demonstraram maior eficiêncialuminescente quando dopadas com concentração de 5% do complexo precursor [Eu(tta)3(TOPO)2]. Estas nanopartículas foram funcionalizadas com 1,6 hexodiamino eposteriormente foram ligadas a um dialdeído que servirá como ponte na reação comas entidades biológicas. No entanto, devido à transferência de energia do sistemaorgânico Ac-Ac/nanossensor, as nanopartículas poliméricas luminescentes de PHBdemonstraram maior intensidade de sinal para a detecção do 17-β-estradiol por espectrofluorimetria. Pode-se concluir que as nanopartículas produzidas e testadasneste trabalho são fortes candidatas para a detecção do 17-β-estradiol em plasma sanguíneo ou outras entidades biológicas, apresentando opções diagnósticas, conduta extremamente importante para direcionamento terapêutico. / Tese (Doutorado em Tecnologia Nuclear) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

A bacia do rio Corumbataí localizada na região central do estado de São Paulo, abrange parte dos municípios de Analândia, Corumbataí, Santa Gertrudes, Rio Claro, Itirapina, Ipeúna, Charqueada e Piracicaba, durante seu percurso os rios: Passa Cinco, Ribeirão Cabeça e Ribeirão Claro, considerados seus principais afluentes, influenciam significativamente nas características de suas águas. Os testes de toxicidade vêm sendo utilizados e reconhecidos mundialmente como uma ferramenta eficaz no biomonitoramento e na avaliação da qualidade das águas e dos sedimentos. Visando avaliar e entender o decréscimo da qualidade da água e do sedimento do rio Corumbataí, após a afluência das águas do Ribeirão Claro foram analisadas amostras em cinco pontos de coleta: dois no Ribeirão Claro, dois no rio Corumbataí e um no rio São Joaquim. Foram realizados testes ecotoxicológicos com Hydra attenuata, Pseudokirchneriella subcapitata, Chironomus xanthus, Lactuca sativa e Dapnhia magna, determinações físicas, químicas e biológicas para avaliação do IQA e quantificação dos elementos químicos: Be, Al, Na, Mg, Ca, K, V, Mn, Fe, Cr, Co, Ni, Cu, Zn, As, Se, Ag, Cd, Sb, Ba, Hg, Pb, Th, Tl, U por Espectrofotometria de massas com fonte de plasma induzido (ICP-MS). As coletas ocorreram trimestralmente de Abril de 2006 a Maio de 2007. Os resultados das quantificações dos elementos químicos foram comparados aos limites determinados pela Resolução CONAMA 357/05, e observados valores de Al, Fe, Hg e Pb, acima do recomendado. As análises físicas, químicas e biológicas mostraram que o oxigênio dissolvido (OD), fósforo total, nitrogênio amoniacal, demanda bioquímica de oxigênio (DBO5dias) e os coliformes fecais estão fora dos padrões recomendados. A avaliação do IQA aponta Classificação Ruim para todas as amostras do rio São Joaquim, para o Ribeirão Claro três Ruins, uma Boa e uma Imprópria, mostrando a importância destes afluentes para a piora das águas do rio Corumbataí. Todas as amostras de água e sedimento apresentaram toxicidade para pelo menos um dos organismos utilizados, principalmente para H. attenuata, P. subcapitata e C. xanthus. A sensibilidade da H. attenuata esteve diretamente relacionada à presença dos coliformes fecais, Cu, Ag, Ni e Zn, enquanto que a da P. subcapitata esteve relacionada à presença de Zn, Na, DBO e diminuição do OD, segundo as análises estatísticas de redundância (RDA) rodadas pelo R estatístico. Os elementos químicos Al e Fe estiveram presentes nas amostras de água e sedimento, acima do recomendado pelo CONAMA para rios de Classe 2, assim como o Mn, o qual teve sua concentração elevada pelas chuvas, enquanto que os elementos Be, V, Cr, Co, Ni, As, Cu, Cd, Sb, Ba, Pb, Zn e U estiveram abaixo ou próximo aos limites. O sedimento apresentou os elementos biodisponível na seguinte ordem decrescente: Mn > Zn > Ba > Pb > Cu > V > Cr > Co > Ni > As > U > Be > Ag > Cd > Se > Hg > Sb, quando retirado o Al e o Fe. Os rios São Joaquim e Ribeirão Claro influenciaram negativamente a qualidade da água do rio Corumbataí / The Corumbataí river basin located on Central region in São Paulo State is composed by part of Anâlandia, Corumbataí, Rio Claro, Itirapina, Ipeúna, Charqueada and Piracicaba municipality. The principals affluents rivers are Passa Cinco, Ribeirão Cabeça and Ribeirão Claro that have influence on water characteristics of Corumbataí river. The toxicity test has been recommended as an efficient tool for biomonitoring and evaluation of water and sediment quality. With the aim of evaluate and understand the decrease of water and sediment qualities of Corumbataí river five sampling sites were selected: two on Ribeirão Claro river, two on Corumbataí river and one on São Joaquim river. Ecotoxicity tests were performed with Hydra attenuata, Pseudokirchneriella subcapitata, Chironomus xanthus, Lactuca sativa and Dapnhia magna. For evaluation o IQA, were carried out physical, chemical and biology determinations as well chemical elements quantification by ICP-MS, such as: Be, Al, Na, Mg, Ca, K, V, Mn, Fe, Cr, Co, Ni, Cu, Zn, As, Se, Ag, Cd, Sb, Ba, Hg, Pb, Th, Tl and U. The water samples were collected every three mouth from April 2006 to May 2007. The chemical elements quantification results were compared with values standardized by CONAMA 357/05 Resolution. The Al, Fe, Hg e Pb concentration were over the ones recommended. The physical, chemical and biology assays showed that the dissolved oxygen, total phosphorus, ammonia nitrogen, biochemical oxygen demand and fecal coliforms were out of the standard recommendation. The IQA evaluation classified all water samples from São Joaquim River as Bad, the Ribeirão Claro three water samples were Bad, one Good and another Improper. So it showed the importance of these affluents on the decrease of Corumbatai river water quality. All the water and sediment samples showed toxicity at least for one organism tested, mainly for H. attenuata, P. subcapitata e C. xanthus. The H. attenuata sensity were directly affected by fecal coliforms, Cu, Ag, Ni and Zn, meanwhile the P. subcapitata were affect by Zn, Na, DBO and lowest level of OD, as obtained by redundancy analysis (RDA) on R statistics program. The chemical elements Al and Fe were present on water and sediment sample, over the recommend concentration of CONAMA 357/05 Resolution for rivers of Class 2 as the same for the Mn, which had high concentration after rain. On the other hand the elements Be, V, Cr, Co, Ni, As, Cu, Cd, Sb, Ba, Pb, Zn and U were below or near the limits. The sediment showed the chemical elements in this order: Mn > Zn > Ba > Pb > Cu > V > Cr > Co > Ni > As > U > Be > Ag > Cd > Se > Hg > Sb, when the Al and Fe where not considered. The São Joaquim and Ribeirão Claro rivers had a negative influence on Corumbataí river water quality

M.Sc. (Zoology) / National water quality monitoring in South Africa has in the past mainly focused on measuring physical and chemical variables. However, it is increasingly realised that measuring physical and chemical variables on their own cannot provide an accurate account of the general "health" of an aquatic ecosystem. Biological communities, on the other hand, are accurate indicators of overall environmental conditions. Water quality management must, therefore, rely on comparative data for both chemical composition and biological effects. In fact, it appears as if biological monitoring (biomonitoring) is worldwide becoming a primary tool in assessing environmental condition and verifying compliance with effluent discharge. This study classified different biomonitoring approaches and techniques under bioassessment (referring to the field oriented biomonitoring protocols which make use of biotic indices to assess water quality); bioassays (toxicity tests which is usually laboratory-based); behavioural bioassays (including aspects such as early warning systems, and preference and avoidance studies); bacteriological studies (the monitoring of certain microbes to allow the detection of faecal contamination); measurement of bioaccumulation (referring to the methods by which the uptake and retention of chemicals in the body of an organism can be monitored); and fish pathology (fish health studies dealing with the causes, processes and effects of disease). Habitat assessment and evaluation was identified as an essential part of any biosurvey. There can be little uncertainty about the mutual dependence of habitat quality, biological health and chemical characteristics of water in the environment. Relative habitat condition, as the principal determinant of attainable biological potential, should set the context for interpreting the results of a biosurvey and can be used as a general predictor of biological condition. Chemistry can further help to explain and characterise certain impacts. The Crocodile River, Eastern Transvaal, was selected for conducting a case study. The SASS2 rapid bioassessment protocol, as well as a habitat quality index (Hal) was used during five consecutive biosurveys. From the results obtained in this study, it appears as if biomonitoring can be used to good effect in overall environmental assessment. The SASS2 index appeared to be both a robust and sensitive indicator of environmental condition. Application of the SASS2 technique on a regional or even national basis should be feasible with regard to simplicity and practicality. It is also cheaper and less labour intensive than comprehensive chemical monitoring. However, bioassessments should not replace but rather compliment chemical and physical monitoring.

L’estuaire de Seine est l’un des plus pollués de l’hémisphère nord dû notamment à ses activités urbaines et agricoles de son bassin versant. Malgré cela le copépode E. affinis représente 90 % de la biomasse zooplanctonique en estuaire de Seine. L’objectif de la thèse était de développer un nouveau Bio-essAi Survie Croissance copépode (BASIC) à l’aide du copépode E. affinis sur différentes matrices environnementales. Dans un premier temps, le bio-essai a été mise au point à l’aide de sédiments enrobés avec des molécules modèles représentatives de la contamination estuarienne (APs, HAPS et PCBs) avant d’être validé, par la suite, sur des sédiments naturels de l’estuaire de Seine. Le bio-essai à ensuite été appliqué en solution au 4-NP et au PCB 126 et sur Vibrio anguillarum issus de l’estuaire de Seine. Un protocole d’exposition multi-générationnel a été appliqué dans les mêmes conditions pendant trois générations. Les résultats recensés ont montré un effet sur le développement larvaire lors des expositions après 6 jours d’expositions à des sédiments enrobés. Dans le cadre des expositions en solution, des résultats similaires ont été obtenus, notamment, un retard de croissance après deux jours d’exposition au 4-NP et au PCB 126 corrélé avec les niveaux d’activité chitobiase. En revanche, à la fin de l’expérimentation ce résultat n’est plus observé. Lors des expositions à Vibrio anguillarum, une augmentation de l’activité chitobiase a été enregistrée les premiers jours de l’exposition, impliquant une augmentation de la croissance au sixième jour. Dans le cadre des expérimentions multi-générationnelle, le copépode semble se stabiliser à la troisième génération. / Seine estuary is one of the most polluted in the northern hemisphere largely due to its urban and agricultural activities of its watershed. Despite this the copepod E. affinis represents 90% of zooplankton biomass in the Seine estuary. The aim of the thesis was to develop a new bioassay Survival Growth copepod with the copepod E. affinis on various environmental matrices. Initially, the bioassay was developed using sediments spiked with molecules representative models of estuarine contamination (APs, PAHS and PCBs) before being validated on natural sediments of the Seine estuary. The bioassay was applied in solution 4-NP and PCB 126 and Vibrio anguillarum from the Seine estuary. A multi-generational exposure protocol was applied under the same conditions for three generations. The results showed an effect on larval development after 6 days of exposure to sediments. In solution, similar results were obtained, including, retarded growth after two days of exposure to 4-NP and PCB 126 correlated with chitobiase activity levels. However, at the end of the experiment the result is not observed. At exposition with Vibrio anguillarum, increased activity was recorded chitobiase the first days, involving an increase in growth in the sixth day. As part of the multi-generational experimenting, the copepod appears to be stabilizing at the third generation.

Abstract Bone morphogenetic proteins (BMPs) constitute a large family of osteoinductive proteins. Different BMPs are widely used in animal experiments and increasingly in the field of bone surgery. However, the sterilization of BMPs and the choice of a suitable mode of delivery, which binds and slowly releases BMP molecules, are still under intensive investigation. The aims of this study were to evaluate the effects of ethylene oxide and gamma sterilizations and different delivery systems on the osteoinductivity of reindeer BMP extract by using heterotopic and orthopic animal models. Ethylene oxide gas (Steri-Vac 4XL, temperature 29 °C, exposure time 4 h, concentration 860 mg/l) and gamma (doses of 3.15 or 4.15 Mrad) sterilizations were used. The tested delivery systems for reindeer BMP were collagen (Lyostypt®), gelatine capsule (no.1) and composites containing collagen combined with tricalcium phosphate (TCP) or hydroxyapatite (HA) or biphasic tricalcium phosphate-hydroxyapatite (TCP/HA) or biocoral (NC) frames. The injectability of reindeer BMP was tested by using injections containing a saline or gelatine vehicle. Osteoinductivity was evaluated as ectopic bone formation in the thigh muscle pouches of mouse hind legs. The induced new bone was evaluated based on the incorporation of 45Ca or calcium yield, radiographs and histological examination three weeks after the operations. The effect of gamma sterilization on the bone healing capacity of reindeer BMP extract was evaluated in a rabbit radial bone defect model in comparison with non-sterilized reindeer BMP extract and recombinant BMP-2. Bone healing was evaluated after eight weeks based on radiographs, mechanical tests and peripheral computerized tomography (pQCT). All BMP implants induced new bone in vivo visible in radiographs, but no bone formation was seen in the control implants without reindeer BMP. Gamma sterilization did not decrease significantly the osteoinductivity of reindeer BMP extract, except when administered as an injection containing gelatine vehicle. Ethylene oxide sterilization decreased significantly the osteoinductivity of reindeer BMP extract and was significantly inferior compared to gamma sterilization. Reindeer BMP combined with collagen or composite containing collagen and TCP/HA frame induced new bone significantly better than reindeer BMP combined with composite containing collagen and TCP frame. BMP injections with gelatine or saline vehicles induced new bone effectively. Injections were easy to handle and well tolerated by the mice. Reindeer BMP extract administered with collagen carrier healed the bone defect of the rabbit radius significantly better than control implants without reindeer BMP or no treatment and its effect was comparable with rhBMP-2 treatment.

PhD. (Chemistry) / This thesis focuses on functionalising the silicone membrane by filling it with synthesised γ-Fe2O3 nanoparticles for the purpose of using it as a passive sampling device for monitoring microcystins and cylindrospermopsin in aquatic environments. The identity and purity of the synthesised nanoparticles was verified by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscope (SEM), surface zeta potential analyser and Brunauer-Emmet-Teller (BET). There was no chemical uptake of microcystin compounds into the silicone membrane without a suspension of maghemite nanoparticles. But, a linear uptake of microcystin compounds was observed into the functionalised silicone membrane passive sampler throughout the exposure period. These findings suggest that the silicone membrane filled with a suspension of synthesised γ-Fe2O3 nanoparticles can be an efficient, green and less costly instrument useful in estimating biotoxin concentrations in South African waters and the world at large.

This thesis examined the antimicrobial activity of select neotropical plants from Costa Rica and traditional Q’eqchi Maya medicines from Belize. In particular the potential for interference with bacterial quorum sensing (QS) and biofilm formation as well as fungal growth were assessed. Of one hundred and twenty six extracts collected from Costa Rica, one third showed significant QS inhibition while 13 species displayed more biofilm inhibitory activities than the positive control allicin. The active species belonged to the Lepidobotryaceae, Melastomataceae, Meliaceae, Sapindaceae, and Simaroubaceae. Twelve Marcgraviaceae species were tested for the same biological activities; of these, three showed similar QS inhibition to that of the positive control Delisea pulchra (Greville) Montagne and five with at least 30% biofilm inhibition. Only one species inhibited fungal growth – Marcgravia nervosa Triana & Planch. Bioassay-guided isolation of this plant resulted in the identification of the active principle as a naphthoquinone, with a minimum inhibitory concentration (MIC) ranging from 85 to 100 μM against Saccharomyces cerevisiae. Similarly, sixty one Q’eqchi’ Maya medicinal plant species were evaluated for their antimicrobial activities. Of these, four species showed more QS inhibition than D. pulchra, seven with comparable biofilm inhibitory activities that of allicin, and two with similarly antifungal activity to berberine. Two spirostanol saponins were isolated from Cestrum schlechtendahlii G.Don, an active antifungal plant. The major saponin showed growth inhibition against Saccharomyces cerevisiae and Fusarium graminearum, with MICs of 16.5 μM and 132 μM, respectively. Further analyses of this compound using chemical genomics suggested that its antifungal mechanism of action is pleiotropic, affecting multiple targets. Taken together, these findings showed that neotropical plants and traditional Q’eqchi’ Maya medicines contain phytochemicals that interfere with bacterial biofilm formation and quorum sensing as well as fungal growth.

M.Sc. / The water quality in the Vaal River system has deteriorated due to increasing industrial, mining and agricultural activities in the Vaal Dam and Vaal River Barrage catchment areas. This study was undertaken to determine the water quality in the Vaal Dam, and three localities within the Vaal River Barrage reservoir (i.e. the Riviera Aquatic Club, Abrahamsrust Pleasure Resort and at the Barrage structure). The objectives of this study, was to determine the fish population species composition, evaluate the general health of the selected fish species and obtain site specific metal bioaccumulation data for each locality. Suggestions and proposals for future monitoring and management of these waterbodies were also made. Metereological information on the Vaal River Barrage area was supplied by Rand Water, as well as physical and chemical data of fixed localities, sampled fortnightly (January 1997 to September 1998). In addition, water samples were collected at the Vaal Dam and the three Vaal River Barrage localities mentioned above, during February, May, August and November 1997. These samples were analysed by the Chemistry Department of the Rand Afrikaans University in accordance with standard methods. The chemical and physical characteristics and metal concentrations (aluminium, cadmium, calcium, copper, iron, magnesium, manganese, nickel, strontium and zinc) of these separate sets of data were then compared with Rand Water's Raw Water Quality Criteria. The aluminium, ammonia, chromium, copper, dissolved organic carbon, fluoride, iron, lead, manganese, nitrate, sulphate, total hardness and turbidity variables did not comply with the recommended guideline values. No guideline levels for strontium are available, however. Sedimentation, turbidity, salination and eutrophication within the Vaal River system play an important role in the bioaccumulation of metals by benthic organisms. Metals that adsorb to sediment particles, through various processes, are generally more persistent and less mobile than those in the overlying water and pose a severe long-lasting threat to the aquatic environment. Generally, the highest metal concentrations (AI, Cd, Cu, Fe, Mn, Ni, Pb, Sr and Zn) in the sediment samples collected at the four localities and seasons, were found in the finer sediment classes (class D). The Riviera Aquatic Club locality had the highest overall concentrations of the metals analysed. The adult fish communities within the Vaal Dam and Vaal River Barrage were assessed, as it is believed that the fish community structure is indicative of environmental conditions. Adult Labeo capensis, Labeo umbratus, Cyprinus carpio, Clarias gariepinus, Barbus aeneus and Micropterus sa/moides, were collected (using gill nets) to determine the adult fish community composition and age. When compared with a study conducted by Mulder (1971), it is clear that Labeo capensis has remained the most abundant species within the system, but that Barbus aeneus has now been replaced by Labeo umbratus as the second most abundant species. Furthermore, age determination tables (Mulder, 1971) were used to classify the fish population between the ages of 2 and 7 years. This indicates a healthy, dynamic community structure where recruitment is taking place. The Health Assessment Index (HAl) was used to determine the general health of the two fish species selected for metal analysis (i.e. Labeo capensis and Labeo umbratus). Except for moderate to severe gill damage recorded, no damage to external variables, were of concern. However, both Labeo capensis and Labeo umbratus had high percentages (89% and 93%, respecii tively) of abnormal livers. Using the Plascon Colour Chart (Watson, personal communication), these livers were classified as ranging from tan to "coffee with cream" in colour. High haematocrit and plasma protein levels were observed and could be due to environmental stress, while the White Blood Cell Counts were within normal ranges. Generally speaking, only mild to moderate parasite infections were observed and probably do not pose a serious health threat to the fish host. Total HAl values for Labeo capensis indicate that this this fish population was in relatively good health, but too few Labeo umbratus individuals were collected to make accurate assumptions about this population. Metal analysis (AI, Cd, Cu, Fe, Mn, Ni, Pb, Sr and Zn) was carried out on the tissues and organs (skin, muscle, gill filaments, gill arches, liver, hindgut content, gonads and vertebrae) of Labeo capensis and Labeo umbratus, collected during the study period. Only aluminium, iron and strontium are discussed. Aluminium concentrations were relatively high at all of the localities, but as pH values were mostly alkaline, aluminium is rendered biologically unavailable and as for iron, was mostly accumulated in the non-edible portion and viscera of the fish. Strontium accumulates in bony tissues due to its similarity with calcium, but is not essential for bone formation. The human health risks of these metals in the edible portion of the fish still have to be refined and communicated to the general public of South Africa. In conclusion, this baseline study has determined the metal concentrations in the aquatic environment (due to anthropogenic activities) that may be accumulated by fish and may affect the adult fish community structures and their general health.

>Magister Scientiae - MSc / The high rate of rediscovery of known compounds has led to a decline in the discovery of novel natural products. The high biodiversity of organisms growing in extreme conditions such as oceans has led to the increased interest by researchers for their use as a source of novel natural products. Marine bacteria are known for their extensive biosynthetic capacity to produce diverse natural products, which are suitable for various biotechnology applications such as in agriculture, for treatment of fungal plant pathogens, and as antibiotics, for treatment of bacterial infections. This study aimed at discovering novel secondary metabolites from marine bacteria previously associated with novel marine invertebrate species endemic to the South African coast. The methodologies used in this study included a bioassay guided fractionation coupled to genome sequencing and mining. For the bioassay guided fractionation approach, the study first focused on screening marine bacteria for antimicrobial activity when cultured on 4 different media, against fungal strains previously shown to be virulent olive trunk pathogens. In parallel, the bacterial isolates with the most inhibitory activity against the fungal pathogens were also screened for antimicrobial activity against 4 indicator strains including Gram-negative Escherichia coli 1699 (E. coli), Pseudomonas putida, and Gram-positive Staphylococcus epidermidis ATCC14990, and Bacillus cereus ATCC10702. One of the marine bacterial isolates, PE6-126, showed diverse antimicrobial activity including antibacterial and antifungal activity against the tested strains. The genome sequencing data revealed that this isolate was B. cereus based on the average nucleotide identity (ANI) (>99%) to reference strains. antiSMASH analysis of the genome revealed nine predicted secondary metabolite clusters including bacteriocins (2), non-ribosomal peptide synthetase (NRPS) (2), siderophore (1), sactipeptide (1), betalactone (1), linear azol(in)e-containing peptides (LAP) - bacteriocin (1) and a terpene (1). Some of these pathways had low to no sequence similarity to known pathways, indicating the potential of these pathways to produce novel compounds. One of the pathways showed very high sequence similarity to the thuricin CD pathway in Bacillus thuringiensis. Considering that thuricin CD has been reported to have antimicrobial activity against B. cereus (ATCC1072), it was hypothesised that it could also be produced by PE6-126. However, the antimicrobial extract from PE6-126 was tested for sensitivity to proteinase K and heat treatment, which thuricin CD is known to be sensitive to. The results revealed that the antimicrobial activity was not lost after treatment, implying that a different metabolite could be responsible for the anti-B. cereusactivity. In addition, PE6-126 initially displayed antimicrobial activity against a multi-drug resistant E. coli 1699, suggesting some of the antimicrobial compound/(s) produced by this strain could potentially be novel. The bioassay-guided fractionation approach coupled to Liquid Chromatography Mass Spectrometry (LC-MS) did not lead to identification of the antimicrobial compound/(s), therefore it remains a question whether the secondary metabolite pathways predicted by antiSMASH lead to the production of the active compound/(s).The results from this study showed that even well studied species have the potential to synthesize as yet undescribed compounds, based on the novelty of some of the pathways. This study highlights the importance of employing a genome-guided approach in drug discovery, as there may be many novel compounds to discover from biosynthetic pathways that have not yet been characterised. Further research is needed to identify the antimicrobial compound/(s) produced by PE6-126.

Philosophiae Doctor - PhD / bioassay guided isolation approach was then used to isolate the high molecular weight antibacterial compound (50kDa-100kDa) from T. actiniarum fermentations. With common protein isolation, purification and detection methods failing to provide insight into the nature of the antibacterial compound, we hypothesized that the active agent is not proteinaceous in nature and may be a high molecular weight exopolysaccharide. Extraction and antibacterial screening of the exopolysaccharide fraction from T. actiniarum showed antibacterial activity as well as lytic activity when subjected to a zymography assay using Pseudomonas putida whole cells as a substrate. Additionally, the biosynthetic pathways for the production of poly-β-1, 6-N-acetyl-glucosamine (PNAG), an exopolysaccharide involved in biofilm formation and chondroitin sulfate, a known and industrially important glycosaminoglycan with antibacterial and anti-inflammatory activity was identified and the mechanism may be novel. Genome mining identified a variety of novel secondary metabolite gene clusters which could potentially encode other novel bioactivities. Therefore a bioassay guided isolation, focused on the small (<3kDa) molecules, was pursued. Secondary metabolites were extracted, fractionated and screened for biofilm inhibition, antibacterial and anticancer activity and activity was observed in all assays. Active fractions were dereplicated by UHPLC-QToF-MS and compounds of interest were isolated using mass guided preparative HPLC. The purity of the isolated compounds was assessed using UHPLC-QToF-MS and NMR and the structure of the target compounds elucidated. Structures that could be determined were the bile acids cholic acid and 3-oxo cholic acid and although not responsible for the observed activities, this is the first report of bile acid production for this genus. This is the first study investigating the bioactive potential of the strain and the first demonstrating that T. actiniarum is a promising source of potentially novel pharmaceutically relevant natural products depicted through both culture-dependent and culture-independent approaches.