Drugs that are preferentially absorbed through the stomach or the small intestine have a narrow time window for absorption since passage through this region of the gastrointestinal tract is rapid. A drug delivery system that can adhere to the gastric epithelium will substantially slow down drug transit and help overcome this problem. To achieve this, this study proposes the novel use of a glycan-binding adhesion protein from Helicobacter pylori, BabA, to create targeted drug delivery vectors that can mimic the attachment of this bacterium to the gastric epithelium. In this work, a recombinant form of BabA was expressed in the periplasmic space of Escherichia coli; it was found that after the incorporation of a C-terminal hexa-lysine tag, the expression and purification of this protein was significantly improved to amounts that enabled its subsequent characterisation and application. Recombinant BabA retained the highly selective glycan-binding properties of H. pylori and next, its crystal structure was solved in the absence and presence of Lewisb – a glycan well studied for its role in serving as a receptor for BabA. The structural models revealed that Lewisb binding occurred through a network of hydrogen bonds within a single, shallow binding pocket at the tip of a β-unit in BabA. Binding studies then confirmed that this site was also responsible for the recognition of other glycan receptors. Using this insight, recombinant BabA was conjugated to model drug delivery vectors via a linkage that favoured exposure of its glycan-binding β-unit; the binding properties of BabA successfully translated to these model BabA-vectors. The research presented in this thesis lays a strong foundation for future work to assess the in vitro and in vivo efficacy of biomimetic BabA drug carriers.

Morphological and phylogenetic diversity of 143 strains belonging to Oscillatoriales with focuson traditional genera Phormidium sensu lato and Microcoleus were studied. The 88 strains of Ph. autumnale, Ph. setchelianum, Ph. subfuscum, Ph. favosum etc., and M. vaginatus confirmed the generic identity with typical Microcoleus Desmazi?res ex Gomont. The necessary nomenclatoric transfers were realized defining the revised genus Microcoleus. Based on phylogeny and morphology the taxonomic revision of the Antarctic species Ph. murrayi (Lyngbya murray West & West) was implemented and the genus Wilmottia was established. The phylogenetic evaluation of morpho-species included in Phormidium group I (Ph. lloydianum and Ph. acuminatum Gomont) preceded the definition of species Oxynema thaianum spec. nova. The biogeography of Antarctic and Arctic strains of M. vaginatus (Ph. autumnale) based on 16S rDNA and ITS (internal transcribed spacer of the 16S rDNA -23SrDNA ribosomal operon) sequences and strain's morphology was evaluated. The comparison of polar and non-polar strains indicated that the Antarctic populations of M. vaginatus remained isolated from time of the isolation of the Antarctica from the Gondwana before ~31?45 Ma, whereas the transport of populations within Arctic is relatively frequent even at the present time. It was shown that the polar strains of M. vaginatus from the north and south polar areas were not identical.

The aim of this thesis is to enhance the extracellular electron transfer rates (exoelectrogenesis) in cyanobacteria, to be utilised for photo-bioelectricity generation in biophotovoltaics (electrochemical cell). An initial cross comparison of the cyanobacterium Synechococcus elongatus PCC7942 against other exoelectrogenic cultures showed a hindered exoelectrogenic capacity. Nonetheless, in mediatorless biophotovoltaics, it outperformed the microalgae Chlorella vulgaris. Furthermore, the performance of S. elongatus PCC7942 was improved by constructing a more efficient design (lower internal resistance), which was fabricated with carbon fibres and nitrocellulose membrane, both inexpensive materials. To strategically obtain higher exoelectrogenic rates, S. elongatus PCC7942 was conditioned by iron limitation and CO2 enrichment. Both strategies are novel in improving cyanobacteria exoelectrogenesis. Iron limitation induced unprecedented rates of extracellular ferricyanide reduction (24-fold), with the reaction occurring favourably around neutral pH, different to the cultural alkaline pH. Iron limited cultures grown in 5% and 20% CO2 showed increased exoelectrogenic rates in an earlier stage of growth in comparison to air grown cultures. Conveniently, the cultural pH under enriched CO2 was around neutral pH. Enhanced photo-bioelectricity generation in ferricyanide mediated biophotovoltaics was demonstrated. Power generation was six times higher with iron limited cultures at neutral pH than with iron sufficient cultures at alkaline pH. The enhanced performance was also observed in mediatorless biophotovoltaics, especially in the dark phase. Exoelectrogenesis was mainly driven by photosynthetic activity. However, rates in the dark were also improved and in the long term it appeared that the exoelectrogenic activity under illumination tended to that seen in the dark. Proteins participating in iron uptake by an alleged reductive mechanism were overexpressed (2-fold). However, oxidoreductases in the outer membrane remain to be identified. Furthermore, electroactive regions in biofilms of S. elongatus PCC7942 were established using cyclic voltammetry. Double step potential chronoamperometry was also successfully tested in the biofilms. Thus, the electrochemical characterisation of S. elongatus PCC7942 was demonstrated, implying that the strategies presented in this thesis could be used to screen for cyanobacteria and/or electrode materials to further develop systems for photo-bioelectricity generation.

Master of Science / Food Science Institute / Curtis Kastner / Harmful Algal Blooms (HABs) including Cyanobacteria and other toxic marine biota are responsible for similar harmful effects on human health, food safety, ecosystem maintenance, economic losses and liability issues for aquaculture farms as well as the food industry. Detection, monitoring and mitigation are all key factors in decreasing the deleterious effects of these toxic algal blooms. Harmful algal blooms can manifest toxic effects on a number of facets of animal physiology, elicit noxious taste and odor events and cause mass fish as well as animal kills. Such blooms can adversely impact the perception of the efficacy and safety of the food industry, water utilities, the quality of aquaculture and land farming products, as well as cause ripple effects experienced by coastal communities. HABs can adversely impact coastal areas and other areas reliant on local aquatic ecosystems through the loss of revenues experienced by local restaurants, food manufacturers as well as seafood harvesting/processing plants; loss of tourism revenue, decreased property values and a fundamental shift in the lives of those that are reliant upon those industries for their quality of life. This paper discusses Cyanobacteria, macroalgae, HABs, Cyanobacteria toxins, mitigation of HAB populations and their products as well as the ramifications this burgeoning threat to aquatic/ landlocked communities including challenges these toxic algae pose to the field of food science and the economy.

abstract: Large-scale cultivation of photosynthetic microorganisms for the production of biodiesel and other valuable commodities must be made more efficient. Recycling the water and nutrients acquired from biomass harvesting promotes a more sustainable and economically viable enterprise. This study reports on growing the cyanobacterium Synechocystis sp. PCC 6803 using permeate obtained from concentrating the biomass by cross-flow membrane filtration. I used a kinetic model based on the available light intensity (LI) to predict biomass productivity and evaluate overall performance. During the initial phase of the study, I integrated a membrane filter with a bench-top photobioreactor (PBR) and created a continuously operating system. Recycling permeate reduced the amount of fresh medium delivered to the PBR by 45%. Biomass production rates as high as 400 mg-DW/L/d (9.2 g-DW/m2/d) were sustained under constant lighting over a 12-day period. In the next phase, I operated the system as a sequencing batch reactor (SBR), which improved control over nutrient delivery and increased the concentration factor of filtered biomass (from 1.8 to 6.8). I developed unique system parameters to compute the amount of recycled permeate in the reactor and the actual hydraulic retention time during SBR operation. The amount of medium delivered to the system was reduced by up to 80%, and growth rates were consistent at variable amounts of repeatedly recycled permeate. The light-based model accurately predicted growth when biofilm was not present. Coupled with mass ratios for PCC 6803, these predictions facilitated efficient delivery of nitrogen and phosphorus. Daily biomass production rates and specific growth rates equal to 360 mg-DW/L/d (8.3 g/m2/d) and 1.0 d-1, respectively, were consistently achieved at a relatively low incident LI (180 µE/m2/s). Higher productivities (up to 550 mg-DW/L/d) occurred under increased LI (725 µE/m2/s), although the onset of biofilm impeded modeled performance. Permeate did not cause any gradual growth inhibition. Repeated results showed cultures rapidly entered a stressed state, which was followed by widespread cell lysis. This phenomenon occurred independently of permeate recycling and was not caused by nutrient starvation. It may best be explained by negative allelopathic effects or viral infection as a result of mixed culture conditions. / Dissertation/Thesis / Masters Thesis Civil and Environmental Engineering 2015

abstract: Hydrothermal environments are important locales for carbon cycling on Earth and elsewhere in the Universe. Below its maximum temperature (~73 °C), microbial photosynthesis drives primary productivity in terrestrial hydrothermal ecosystems, which is thought to be performed by bacterial phototrophs in alkaline systems and eukaryotic algae in acidic systems, yet has received little attention at pH values intermediate to these extremes. Sequencing of 16S and 18S rRNA genes was performed at 12 hot springs with pH values 2.9-5.6 and revealed that cyanobacteria affiliated with the genus Chlorogloeopsis and algae of the order Cyanidiales coexisted at 10 of the sites. Cyanobacteria were present at pH values as low as 2.9, which challenges the paradigm of cyanobacteria being excluded below pH 4. Presence of the carotenoid β-cryptoxanthin in only 2 sites and quantitative PCR data suggest that algae were inactive at many of the sites when sampled. Spatial, but perhaps not temporal, overlap in the habitat ranges of bacterial and eukaryal microbial phototrophs indicates that the notion of a sharp transition between these lineages with respect to pH is untenable. In sedimentary basins, biosphere-derived organic carbon is subjected to abiotic transformations under hydrothermal conditions. Benzaldehyde was experimentally evaluated as a model to assess the chemistry of aldehydes under these conditions. It was first demonstrated that gold, a traditional vessel material for hydrothermal experiments, caused catalysis of benzaldehyde degradation. Experiments in silica tubes were performed at 250, 300, and 350 °C yielding time-dependent data at several starting concentrations, which confirmed second-order kinetics. Therefore, disproportionation was expected as a major reaction pathway, but unequal yields of benzoic acid and benzyl alcohol were inconsistent with that mechanism. Consideration of other products led to development of a putative reaction scheme and the time dependencies of these products were subjected to kinetic modeling. The model was able to reproduce the observed yields of benzoic acid and benzyl alcohol, indicating that secondary reactions were responsible for the observed ratios of these products. Aldehyde disproportionation could be an unappreciated step in the formation of carboxylic acids, which along with hydrocarbons are the most common organic compounds present in natural systems. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2016

Dans le lagon de Moorea, en Polynésie Française, nous avons identifié un écosystème constitué de deux producteurs primaires (les cyanobactéries filamenteuses Lyngbya majuscula et Anabaena cf. torulosa), trois mollusques herbivores (Stylocheilus striatus, S. longicauda, et Bulla orientalis), un nudibranche carnivore (Gymnodoris ceylonica) et un crabe carnivore (Thalamita coerulipes). L. majuscula et A. cf torulosa prolifèrent sur de vastes zones jusqu’à épiphyter les coraux ; elles sont des producteurs importants de métabolites secondaires, principalement des lipopeptides cycliques, qui peuvent être toxiques ou répulsifs. Cependant, ces composés n’empêchent pas le lièvre de mer S. striatus de consommer les cyanobactéries. S. striatus, décrit comme un prédateur spécialiste de L. majuscula, est connu pour séquestrer et/ou biotransformer les métabolites secondaires de L. majuscula. Cependant nous avons également observé S. striatus, sur A. cf torulosa où il semble moins exposé à la prédation du nudibranch G. ceylonica que quand il est sur L. majuscula. Dans cet écosystème modèle, nous avons combiné le profilage des métabolomes des deux cyanobactéries et des expériences en écologie dans le but d’étudier le rôle des médiateurs chimiques dans la structuration de cet écosystème ; nous avons complété la caractérisation des profils métaboliques des deux cyanobactéries, étudié les transmissions verticale et horizontale des métabolites secondaires produits par les cyanobactéries le long de la chaine trophique, et étudié le rôle de ces composés dans les relations prédateurs-proies. De A. cf torulosa, nous avons isolé cinq analogues acyliques et deux analogues cyliques des laxaphycines que nous avons caractérisés par RMN (1D et 2D RMN : COSY, TOCSY, HSQC, HMBC, NOESY), spectrométrie de masse (spectrométrie de masse à haute résolution et fragmentation en MSn), ainsi que par dégradation chimique avec la méthode de Marfey. La présence de laxaphycines acycliques n’a jamais été décrite auparavant. Nous avons montré que les peptides de L. majuscula sont séquestrés sans biotransformation par les herbivores, alors que les herbivores présents sur A. cf torulosa biotransforment deux laxaphycines en quatre composés nouveaux que nous avons caractérisés. Il ne semble pas que la séquestration et la biotransformation soient opérées dans le but d’améliorer les défenses chimiques des herbivores mais plutôt comme un mécanisme de tolérance. Nous avons également montré que les mollusques herbivores utilisent les composés produits par les cyanobactéries comme signaux chimiques pour détecter à distance les cyanobactéries et pour le choix de leur nourriture. Ces expériences de choix semblent indiquer que S. striatus et B. orientalis sont des herbivores généralistes bien que l’influence des molécules des cyanobactéries suggère un comportement adaptatif permettant au mollusque de retrouver l’hôte sur lequel il a été prélevé. / In the lagoon of Moorea in French Polynesia, we have identified a relatively simple tropical marine ecosystem consisting of two primary producers (two filamentous cyanobacteria, Lyngbya majuscula and Anabaena cf. torulosa), three herbivorous molluscs (Stylocheilus striatus, S. longicauda and Bulla orientalis), a carnivorous nudibranch (Gymnodoris ceylonica) and a carnivorous crab (Thalamita coerulipes). L. majuscula and A. cf torulosa, that bloom ephemerally across wide sandy areas and even on corals, are prolific producers of secondary metabolites, mainly cyclic lipopeptides, which may either be toxic or act as feeding deterrents to potential consumers. However, these compounds do not prevent the sea hare S. striatus, feeding on cyanobacteria. S. striatus, considered as L. majuscula specialist, is known to sequester and transform some secondary metabolites produced by L. majuscula,. However we found also S. striatus feeding on A. cf torulosa and in this case it was less susceptible to predation by the nudibranch G. ceylonicasa than when it fed on L. majuscula. In the study of this model ecosystem, we combine cyanobacterial metabolome profiling and ecological bioassays in order to study the cascading effects of chemical mediators in multi-trophic relationships; we completed the metabolic profile characterization of the two cyanobacteria, we studied vertical and horizontal transmissions of the cyanobacterial secondary metabolites along the trophic web, and studied the role of these compounds in predator-prey relationships. Focusing our attention on A. cf torulosa we isolated seven new lipopeptides, derived from the known laxaphycins, and characterized them using extensive NMR experiments (1D and 2D NMR: COSY, TOCSY, HSQC, HMBC, NOESY), mass spectrometry (HR-MS and fragmentation by MSn) and Marfey’s advanced method. It is the first time that acyclic analogs of laxaphycins have been described. Although the peptides from L. majuscula are found intact in herbivores, some lipopeptides from A. cf torulosa are biotransformed by sea hares into four new compounds we characterized. The sequestration and biotransformation by the herbivores may be considered as a tolerance mechanism rather than a defense mechanism. We demonstrate also that the herbivores use cyanobacterial compounds as chemical cues for cyanobacteria tracking and feeding choice. Our experiments suggest that S. striatus and B. orientalis are generalist consumers, although the influence of cyanobacterial chemical cues on their foraging preferences may suggest an adaptive behavior enabling the mollusc to track their host of origin.

Submitted by LEILA DOS SANTOS null (leila_snt@hotmail.com) on 2016-08-02T15:43:06Z No. of bitstreams: 1 Leila_dissertacao_oficial.pdf: 2340865 bytes, checksum: b4f4ed59eacc2ee741e0e082a9417b01 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-03T16:55:57Z (GMT) No. of bitstreams: 1 machado_ls_me_soro.pdf: 2340865 bytes, checksum: b4f4ed59eacc2ee741e0e082a9417b01 (MD5) / Made available in DSpace on 2016-08-03T16:55:57Z (GMT). No. of bitstreams: 1 machado_ls_me_soro.pdf: 2340865 bytes, checksum: b4f4ed59eacc2ee741e0e082a9417b01 (MD5) Previous issue date: 2016-06-16 / Em geral, é comum que os reservatórios no Brasil apresentem características propensas a eutrofização, constituindo ecossistemas favoráveis para a expansão das florações de algas e de cianobactérias potencialmente tóxicas. O reservatório de Guarapiranga é responsável pelo fornecimento de água para grande parte da região metropolitana de São Paulo (RMSP), porém, nos últimos anos, tem sido observada uma aceleração do processo de eutrofização mediante interferência humana. Com a alteração das características físico-químicas da água, florações tornaram-se cada vez mais frequentes. Assim, este estudo objetivou investigar a interação e resposta da comunidade fitoplanctônica às variáveis físico-químicas da água do reservatório, bem como, analisar a variação espacial e temporal da comunidade fitoplanctônica, buscando conhecer a dinâmica das cianobactérias e a presença de microcistina. Para isso, foram coletadas amostras de água no reservatório no período chuvoso e seco na região à montante, intermediária (tributário Parelheiros) e barragem. Em laboratório, foram analisadas as características físico-químicas, quantificação de microcistina, triagem e identificação taxonômica dos organismos fitoplanctônicos. Por meio da análise descritiva e estatística foi possível constatar que composição da comunidade fitoplanctônica é diferente ao longo do reservatório e varia em resposta às características físico-químicas de cada local. Em geral, o período de seca representou um fator agravante do processo de eutrofização no reservatório, principalmente na região da barragem, onde foi observado um aumento considerável na biomassa fitoplanctônica. Na região de Parelheiros, foi observada a dominância de cianobactérias potencialmente tóxicas (Cylindrospermopsis raciborskii, no período chuvoso e Planktolyngbya limnetica, no período de seca) e a presença de microcistina, associadas às elevadas concentrações de nutrientes. Desse modo, foi constatado que a comunidade fitoplanctônica responde à variação temporal e espacial observada para as variáveis físico-químicas. De maneira geral, nutrientes como o amônio, nitrogênio e fósforo total estiveram relacionados à alta biomassa fitoplanctônica, principalmente de cianobactérias potencialmente tóxicas. / In general, it is common that the reservoirs in Brazil have the characteristics prone to eutrophication, constituting favorable ecosystem for the expansion of algal blooms and potentially toxic cyanobacteria. The Guarapiranga reservoir is responsible for providing water for a large proportion of the metropolitan area of São Paulo (MRSP), but in recent years it has been observed an accelerated eutrophication process by human interference. By changing the physical and chemical characteristics of water, algal blooms have become increasingly frequent. This study aimed to investigate the composition and biomass of the phytoplankton community as well as the effects of main environmental variables on the phytoplankton community structure, additionally, to analyze the dynamics of cyanobacteria and the quantification of microcystin. Wather samples for phisical, chemical and byologic analysis were collected in the rainy and the dry season in the regions of the reservoir: upstream, intermediate (Parelheiros affluent) and dam. The organisms were analyzed in the microscope, identified and measured for the calculation of the biomass. Statistical analyses indicated that the composition of the phytoplankton community is different along the reservoir and varies in response to the physicochemical characteristics of each site. In general, the dry period represented an aggravating factor in the eutrophication process in the reservoir, mainly in the dam area, where it was observed a observed a significant increase in phytoplankton biomass. In Parelheiros region was observed dominance of potentially toxic cyanobacteria (Cylindrospermopsis raciborskii in the rainy season and Planktolyngbya limnetica, in the dry season) and the presence of microcystin associated with high concentrations of nutrients. Thus, it contacted the phytoplankton community responds to the temporal and spatial variation observed for the physical and chemical variables. In general, nutrients such as ammonia, total nitrogen and total phosphorus were related to high phytoplankton biomass, particularly of potentially toxic cianobactéria.

Photoautotrophic microorganisms, i.e. cyanobacteria and microalgae, are ubiquitous in the glacial ecosystem of the high Arctic archipelago of Svalbard. Their communities play significant roles in the ecosystem, including organic carbon production on the glacier surface and its supply to downstream environments, initiating microbial colonisation after glacier retreat and preparing proglacial substrata for further succession.

This project was focused on development of extraction and purification protocol for novel cytotoxic compound isolated from soil cyanobacterium Nostoc muscorum which would allow to determine its molecular structure via NMR (nuclear magnetic resonance) and MS (mass spectrometry) techniques. Purified cytotoxin was subjected to several biochemical and microscopical experiments in order to assess its toxicological parameters, determine the mechanism of action and evaluate potential application in biotechnology or pharmacy.