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Due to an ever-increasing global population and limited resource availability, there is a constant need for detection of both natural and anthropogenic hazards in water, air, food, and material goods. Traditionally a different instrument would be used to detect each class of contaminant, often after a concentration or separation protocol to extract the analyte from its matrix. Raman spectroscopy is unique in its ability to detect organic or inorganic, airborne or waterborne, and embedded or adsorbed analytes within environmental systems. This ability comes from the inherent abilities of the Raman spectrometer combined with concentration, separation, and signal enhancement provided by drop coating deposition Raman (DCDR) and surface-enhanced Raman spectroscopy (SERS). Herein the capacity of DCDR to differentiate between cyanotoxin variants in aqueous solutions was demonstrated using principal component analysis (PCA) to statistically demonstrate spectral differentiation. A set of rules was outlined based on Raman peak ratios to allow an inexperienced user to determine the toxin variant identity from its Raman spectrum. DCDR was also employed for microcystin-LR (MC-LR) detection in environmental waters at environmentally relevant concentrations, after pre-concentration with solid-phase extraction (SPE). In a cellulose matrix, SERS and normal Raman spectral imaging revealed nanoparticle transport and deposition patterns, illustrating that nanoparticle surface coating dictated the observed transport properties. Both SERS spectral imaging and insight into analyte transport in wax-printed paper microfluidic channels will ultimately be useful for microfluidic paper-based analytical device (𝜇PAD) development. Within algal cells, SERS produced 3D cellular images in the presence of intracellularly biosynthesized gold nanoparticles (AuNP), documenting in detail the molecular vibrations of biomolecules at the AuNP surfaces. Molecules involved in nanoparticle biosynthesis were identified at AuNP surfaces within algal cells, thus aiding in mechanism elucidation. The capabilities of Raman spectroscopy are endless, especially in light of SERS tag design, coordinating detection of analytes that do not inherently produce strong Raman vibrations. The increase in portable Raman spectrometer availability will only facilitate cheaper, more frequent application of Raman spectrometry both in the field and the lab. The tremendous detection power of the Raman spectrometer cannot be ignored. / Ph. D.

Diese Dissertation ist ein Beitrag zum Forschungsfeld der Stressökologie, im Spe-ziellen der Verhaltensökotoxikologie. Das spontane lokomotorische Verhalten der Fischarten Danio rerio und Leucaspius delineatus wurde unter sublethaler Expo-sition mit dem Cyanobakterientoxin Microcystin-LR (MC-LR) und dem Xenobio-tikums 2.4.4`-Trichlorobiphenyl (PCB 28) quantifiziert. Die Schwimmgeschwin-digkeit und Anzahl der Wendungen wurden kontinuierlich mit einem automati-schen Video-Monitoringsystem unter Laborbedingungen aufgezeichnet. In Hin-blick auf zyklische Aspekte wurden die Verhaltensanalysen mit chronobiologi-schen Methoden kombiniert. Hiermit wurde gezeigt, dass MC-LR und PCB 28 zu signifikanten Effekten in Verhalten und Aktivitätsrhythmik beider Fischarten führten. Höhere Konzentrati-onen beider Untersuchungssubstanzen verursachten eine deutliche Aktivitätsredu-zierung bei Danio rerio und Leucaspius delineatus. Einige der festgestellten Do-sis-Wirkungsbeziehungen entsprechen der Hormesistheorie, z. B. war bei geringe-ren MC-LR Konzentrationen ein Aktivitätsanstieg und bei höheren ein Aktivitäts-abfall beider Fischarten zu verzeichnen. Die Exposition mit MC-LR und PCB 28 verringerte bei beiden Testfischarten die Synchronisation der Aktivität mit dem Zeitgeber Licht. Dies führte bei beiden Fischarten zu einer Phasenverschiebung. Bei Leucaspius delineatus war unter dem Einfluss von MC-LR eine Phasenumkehr zu verzeichnen, die Fische wechselten von Tag- zu Nachtaktivität. Die Cosinor Analyse zeigte Dosis abhängige Veränderungen der circadianen Rhythmen der Schwimmaktivität (z.B. MESOR, Akrophase) unter Einfluss von MC-LR und PCB 28 an. Die Power Spektral Analyse indizierte für beide Fischar-ten unter Einwirkung von MC-LR and PCB 28 eine reduzierte Dominanz des cir-cadianen Rhythmuspeaks. Da die registrierten Unterschiede in der Reaktion beider Fischarten auf MC-LR und PCB 28 eher gering waren, sind Ergebnisse der Art Danio rerio, die häufig in Toxizitätstests verwendet wird, mit denen der einheimischen Art Leucaspius deli-neatus vergleichbar. Die Ergebnisse belegen, dass Verhaltensuntersuchungen in Kombination mit chronobiologischen Auswertemethoden eine sensitive und zuverlässige Abschät-zung des Gefährdungspotentials von Substanzen sowohl auf dem Gebiet der Öko-toxikologie als auch für Biomonitoring ermöglichen. / This thesis contributes to the field of stress ecology specifically behavioural ecotoxicology. The spontaneous locomotor behavior of two fish species Danio re-rio and Leucaspius delineatus was recorded and quantified continuously under sublethal exposure to the cyanotoxin microcystin-LR (MC-LR) and the xenobioti-cum 2.4.4`-trichlorobiphenyl (PCB 28). By using an automated video-monitoring system, the swimming velocity and the number of turns were assessed under labo-ratory conditions. For analysing cyclic aspects basic behavioural analyses were combined with chronobiological procedures as cosinor analysis and power spec-tral analysis. Both MC-LR and PCB 28 acted as stressors and caused significant changes in the behaviour and circadian activity rhythms of Danio rerio as well as Leucaspius de-lineatus. So for both species elevated concentrations of the stressors led to a re-duction of their activity. Some dose-responses correspond to the hormesis theory, e.g., there was an increase of daytime activity at lower MC-LR concentrations and a decrease at elevated concentrations of MC-LR for both species. A degree of desynchronisation of activity to the zeitgeber light, which led to a phase shift was caused by the chemicals in both fish species. In Leucaspius de-lineatus this shift was so drastic that this species reversed their significant diurnal activity and became nocturnal under the influence of MC-LR. The Cosinor analysis revealed MC-LR- and PCB 28-induced, dose-dependent al-terations of the circadian rhythms of activity (e.g., MESOR, acrophase). The power spectral analysis showed that the dominance of the circadian rhythmic peak (of 24 h) was reduced under MC-LR and PCB 28 for both species. Since the observed differences in the reactions of both species to MC-LR and PCB 28 were rather small, the results of the species Danio rerio which is widely used for environmental risk assessment tests, are comparable to those of the native European species Leucaspius delineatus. The findings of this study proved that the basic behavioural analyses combined with chronobiological procedures could be valuable tools for the study of stressful or even harmful environmental factors in the field of ecotoxicology as well as for biomonitoring.

Magister Scientiae - MSc / The toxicity of microcystin is associated with the inhibition of serine/threonine protein phosphatases 1 and 2A, which can lead to hepatocyte necrosis and haemorrhage. Analysis of microcystin is most commonly carried out using reversed-phase high performance liquid chromatographic methods (HPLC) combined with ultra-violet (UV) detection .The ability of these techniques to identify unknown microcystin in environmental samples is also restricted by the lack of standard reference materials for the toxins. Highly specific recognition molecules such as antibodies and molecularly imprinted polymers (MIPs) have been employed in the pre-concentration of trace levels of microcystin from water and show great potential for the clean-up of complex samples for subsequent analysis. New biosensor technologies are also becoming available, with sufficient sensitivity and specificity to enable rapid ‗on-site‘ screening without the need for sample processing. In this work we constructed a Protein phosphatase biosensor for detection of microcystin-LR in aqueous medium, onto polyamic acid/graphene oxide (PAA: GO) composite electrochemically synthesised in our laboratory. The composites were synthesised at three different ratios i.e. 50:50, 80:20 and 20:80 to evaluate the effect of each component in the search to produce highly conductive mediator platforms. The electrochemistries of the three different composites were evaluated using CV and SWV to study interfacial kinetics of the materials as thin films at the glassy carbon electrode. The phosphatase biosensor parameters were evaluated using CV, SWV, EIS and Uv-vis spectroscopy. The affinity binding of the microcystin-LR to protein phosphatase 2A was investigated using electrochemical impedance spectroscopy which is a highly sensitive method for measuring interfacial kinetics of biosensor systems.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / This work as main objective to evaluate the eletrogenic effect in preparations of Ãleum of rabbit fixes in chambers of Ãssing, in presence of supernatant of macrophages (S.MfS), stimulated with microcistin-LR (MCLR), of Microcystis aeruginosa. S.MfS estimulated with MCLR (3,2.10-7M; 9,6.10-7M e 3,2.10-6M), produces effect secretion, of the form dose-dependent; being that short circuit current (Isc), the secular chain variation (t) can be described for an equation of the type Isc = a . ekt for a correlation coefficient r = 0,9988 and Iscmaximo = 128,16  14,54 ÂA . cm-2. Later, was observed that the metabolic processes associatesâ geneses of the FSI, from stimulated macrophages, require the participation of a protein G, sensible pertussis active toxin. It was also verified that inhibing of protÃic synthesis, proteases, phosfolipase A2, ciclooxigenases, lipoxigenases, synthesis TNF-a, and antagonists of the PAF, they had reduced the FSI synthesis. With the application of monoclonal antibodies, it was verified that interleukin-b (IL-1b), was the main FSI; as also, that macrophages stimulated with MCLR, in the concentrations above, produced IL-1b and TNF-a, of form dose-dependent. The pay-treatment of the ileum mucosal with bumetanide, indomethacin, tetrodotoxin and HOE, disclosed that the secretory effect, by means of stimulated action of the S.MfS with MCLR is dependent of the secretion of ions chloride, with the participation of PAF, prostaglandins and mediators of the enteric nervous system. With this effect, it associates reduction of the transepitelial resistance (Rte), also mediated for prostaglandins, TNF-a, and indirectly IL-1b. The analysis of the coefficient of Hurst, disclosed that these effect had not occurred of random form, but had involved significant alterations in the kinetic parameters of the eletrogÃnic effect, to the level of the ileum mucosal. Macrophages stimulated for MCLR, produces and liberate more TNF-a of that IL-1b, to the Constant taxes, being this a linked characteristic to the type of employed stimulation. It is concluded, therefore, that MCLR stimulates macrophages to produce substances that can act as intestinal secretion factor, to the level of the ileum mucosal, involving chloride canals, reduction of the Rte, requesting for this, the participation of pro-inflammatory mediators and the enteric nervous system. / Este trabalho teve-se como principal objetivo, avaliar os efeitos eletrogÃnicos em preparaÃÃes de Ãleo de coelho fixadas em cÃmaras de Ãssing, em presenÃa de sobrenadante de macrÃfagos (S.MfS) estimulados com microcistina-LR MCLR de Microcystis aeruginosa. S.MfS estimulados com MCLR (3,2.10-7M; 9,6.10-7M e 3,2.10-6M), produz, de forma dose-dependente; sendo que a variaÃÃo temporal (t) da corrente de curto-circuito (Isc), pode ser descrita por uma equaÃÃo do tipo Isc = a . ekt; para um coeficiente de correlaÃÃo r = 0,9988 e Iscmaximo = 128,16  14,54 ÂÂcm-2. Posteriormente, observou-se que os processos metabÃlicos associados à gÃnese do fator deâ secreÃÃo intestinalâ (FSI), a partir de macrÃfagos estimulados, requer a participaÃÃo de uma proteÃna G sensÃvel à toxina pertusis ativa. Verificou-se tambÃm que inibidores de sÃntese protÃica, proteases, fosfolipase A2, cicloxigenases, lipoxigenases,; sÃntese de TNF-a e antagonista do PAF, reduziram a sÃntese de FSI. Com o emprego de anticorpos monoclonais, verificou-se que IL-1b era o principal FSI; como tambÃm, que macrÃfagos estimulados com MCLR, nas concentraÃÃes acima, reduziu IL-1b e TNF-a, de forma dose-dependente. O prÃ-tratamento da mucosa ileal com bumetanida, indometacina, tetrodotoxina e HOE, revelou que o efeito secretÃrio mediante aÃÃo do S.MfS estimulados com MCLR, à dependente da secreÃÃo de Ãons cloreto, com a participaÃÃo de PAF, prostaglandinas e mediadores do sistema nervoso entÃrico. A este efeito associa-se a diminuiÃÃo da resistÃncia transepitelial (Rte), tambÃm mediada por, prostaglandinas, PAF, TNF-a e, indiretamente, IL-1b. A anÃlise do coeficiente de Hurst (H), revelou que estes efeitos nÃo ocorreram ao acaso, mas envolveram alteraÃÃes significativas nos parÃmetros cinÃticos dos efeitos eletrogÃnicos, ao nÃvel da mucosa ileal. MacrÃfagos estimulados com MCLR produz e libera mais TNFa do que IL-1b, à taxas constantes ; sendo esta uma caracterÃstica atrelada ao tipo de estÃmulo empregado. Conclui-se, portanto, que MCLR estimula macrÃfagos a produzir substÃncias que podem atuar como fator de âsecreÃÃo intestinalâ ao nÃvel da mucosa ileal, envolvendo canais de cloreto, diminuiÃÃo de Rte; requisitando para isto, a participaÃÃo de mediadores prÃ-inflamatÃrios e do sistema nervoso entÃrico.

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