Toxicological effects of a pharmaceutical, simvastatin, in Gammarus locusta: histopathological evaluation

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

Target-specific multiphysics modeling for thermal medicine applications

Dissertation to obtain the degree of Doctor of Philosophy in Biomedical Engineering

Mycetoma fungal infection: multiple organisms as colonizers or pathogens?

We describe a patient with mycetoma or Madura foot, in which histopathological stains of the bone and surface cultures suggested three different organisms including Nocardia species as the cause. Criteria for the diagnosis of the organisms, differentiation between colonizer and pathogen, and significance of mixed infections are discussed.

Toxicity of carcinogenic and non-carcinogenic Polycyclic Aromatic Hydrocarbons and their mixtures to aquatic organisms under ecologically-relevant scenarios

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous priority pollutants that tend to be trapped in aquatic sediments due to their high hydrophobicity. Nonetheless, the differential toxicological effects and mechanisms between the various classes of PAHs and their mixtures, as they invariably occur in the environment, are scarcely known, especially under ecologically-relevant scenarios. This thesis aimed at establishing a bridge between the study of mechanistic pathways and environmental monitoring of carcinogenic and non-carcinogenic PAHs, by introducing ecological-relevance in the research with model PAHs. A first bioassay conducted in situ with the mussel Mytilus edulis demonstrated that, dredging operations in harbours increase PAH bioavailability, eliciting genotoxicity, and showed that established environmental guidelines underestimate risk. Subsequent ex situ bioassays were performed with the carcinogenic benzo[b]fluoranthene (B[b]F) and non-carcinogenic phenantrene (Phe), selected following preceding results, and revealed that low-moderate concentrations of these PAHs in spiked sediments induce genotoxic effects to the clam Ruditapes decussatus, therefore contradicting the general notion that bivalves are less sensitive to PAHs than vertebrates due to inefficient bioactivation. Also, it was demonstrated that passive samplers permit inferring on PAH bioavailability but not on bioaccumulation or toxic effects. On the other hand, sea basses (Dicentrarchus labrax), yielded a complex pattern of effects and responses, relatively to genotoxicity, oxidative stress and production of specific metabolites, especially when exposed to mixtures of the PAHs which led to additive, if not synergistic, effects. It was shown that Phe may elicit significant genotoxicity especially in presence of B[b]F, even though the low, albeit realistic, exposure concentrations diluted dose- and time-independent relationships. The present work demonstrated that environmental quality guidelines underestimate the effects of PAHs in realistic scenarios and showed that the significant genotoxic and histopathological effects caused by mixed PAHs may not be reflected by oxidative stress- or CYP-related biomarkers. Besides important findings on the metabolism of PAH mixtures, the work calls for the need to re-evaluate the criteria for assessing risk and for the disclosure of more efficient indicators of toxicological hazard.

American tripanosomiasis: a study on the prevalence of Trypanosoma cruzi and Trypanosoma cruzi-like organisms in wild rodents in San Luis province, Argentina

INTRODUCTION: Chagas disease is caused by Trypanosoma cruzi. Wild and perianthropic mammals maintain the infection/transmission cycle, both in their natural habitat and in the peridomestic area. The aim of this paper was to present the results from a study on wild rodents in the central and northern regions of San Luis province, Argentina, in order to evaluate the prevalence of this infection. METHODS: Sherman traps were set up in capture areas located between latitudes 32º and 33º S, and longitudes 65º and 66º W. The captured rodents were taxonomically identified and hemoflagellates were isolated. Morphological, biometric and molecular studies and in vitro cultures were performed. Infection of laboratory animals and histological examination of the cardiac muscle and inoculation area were also carried out. Parasites were detected in circulating blood in Calomys musculinus, Graomys griseoflavus, Phyllotis darwini and Akodon molinae. The parasites were identified using biological criteria. Molecular PCR studies were performed on some isolates, which confirmed the characterization of these hemoflagellates as Trypanosoma cruzi. RESULTS AND CONCLUSIONS: Forty-four percent of the 25 isolates were identified as Trypanosoma cruzi, and the remaining 56% as Trypanosoma cruzi-like. These findings provide evidence that wild rats infected with Trypanosoma cruzi and Trypanosoma cruzi-like organisms are important in areas of low endemicity.

Detection of purkinje images for automatic positioning of fixation target and interferometric measurements of anterior eye chamber

In cataract surgery, the eye’s natural lens is removed because it has gone opaque and doesn’t allow clear vision any longer. To maintain the eye’s optical power, a new artificial lens must be inserted. Called Intraocular Lens (IOL), it needs to be modelled in order to have the correct refractive power to substitute the natural lens. Calculating the refractive power of this substitution lens requires precise anterior eye chamber measurements. An interferometry equipment, the AC Master from Zeiss Meditec, AG, was in use for half a year to perform these measurements. A Low Coherence Interferometry (LCI) measurement beam is aligned with the eye’s optical axis, for precise measurements of anterior eye chamber distances. The eye follows a fixation target in order to make the visual axis align with the optical axis. Performance problems occurred, however, at this step. Therefore, there was a necessity to develop a new procedure that ensures better alignment between the eye’s visual and optical axes, allowing a more user friendly and versatile procedure, and eventually automatizing the whole process. With this instrument, the alignment between the eye’s optical and visual axes is detected when Purkinje reflections I and III are overlapped, as the eye follows a fixation target. In this project, image analysis is used to detect these Purkinje reflections’ positions, eventually automatically detecting when they overlap. Automatic detection of the third Purkinje reflection of an eye following a fixation target is possible with some restrictions. Each pair of detected third Purkinje reflections is used in automatically calculating an acceptable starting position for the fixation target, required for precise measurements of anterior eye chamber distances.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor β-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to “in-cell” Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.

Recommendations for Nova School of Business and Economics to target yhe Colombian market

The growing connotation Internationalization has worldwide, alongside the economic, political and socio-environmental changes, is empowering a progressively global education economy. Therefore, this Work Project aims to help Nova SBE to understand the decision making process of the Colombian tertiary education students, as this market constitutes an enriching opportunity to meet both business and educative objectives. In order to do so, a qualitative research was conducted to comprehend the rationale behind Colombian students to study abroad. The study points out that the reputation of a HE institution and the Portuguese culture are the key attributes to pursue a degree in Portugal.

A new and efficient synthesis of benzoimidazopurine derivatives

[Excerpt] Purine nucleobases are essential biomolecules in living organisms. Playing several key roles in the cell, they have been a significant inspiration for drug design.1 Benzimidazole nucleus is an important pharmacophore in the development of molecules with pharmaceutical or biological interest. Benzimidazoles have been reported to display significant pharmacological activities such as antiulcer, antifungal, antiparkinson, anticancer and antibiotic.2 Fused structures incorporating these two scaffolds might be important for medicinal chemistry and, to the best of our knowledge, there are no reports of these systems in the literature. In particular, benzo[4,5]imidazo[2,1]purines seem to be novel and must be important target molecules in the heterocyclic synthesis. (...)

Búsqueda de nuevos extractos y compuestos obtenidos a partir de plantas del centro de Argentina con acción inhibidora de enzimas asociadas a patologías y a la supervivencia de organismos perjudiciales. Search for new extracts and compounds obtained from plants of central Argentina with inhibitoryactivity on enzymes associated with diseases and survival of harmful organisms.

La inhibición en la actividad de ciertas enzimas esenciales puede generar disturbios en la fisiología de algunos organismos como insectos, plantas y microorganismos y en muchos casos puede conducir a su muerte. Por otro lado, la inhibición de estas proteínas logra modificar factores implicados en la manifestación de determinadas enfermedades. Entre las enzimas que muestran estas características podemos mencionar a tirosinasa, p-hidroxifenilpiruvato dioxigenasa (HPPD) y acetilcolinesterasa (AChE). Debido a la necesidad de nuevas drogas con acción inhibidora de las mencionadas enzimas,los investigadores están explorando el mundo vegetal con el fin de obtenerlas,ya que se ha comprobado que las plantas son capaces de sintetizar esta clase de moléculas. Plantas nativas de nuestra región presentan esta propiedad. Continuando con la búsqueda de compuestos bioactivos obtenidos de plantas, se propone en este proyecto obtener nuevos agentes naturales altamente efectivos en inhibir las nombradas enzimas a partir de 100 plantas nativas de la región central de Argentina. Los compuestos aislados pueden ser utilizados directamente o servir como modelo para la síntesis de análogos. En primer lugar se determinará la efectividad de los extractos obtenidos a partir de las plantas seleccionadas como inhibidores de HPPD (utilizando el método del enol-borato) con el fin de seleccionar el más potente. A partir de este extracto y de aquellos seleccionados como más potentes en inhibir tirosinasa y AChE se aislarán, mediante aislamiento bioguiado, e identificarán el/los compuesto/s responsables. Este proceso será llevado a cabo por técnicas cromatográficas y espectroscópicas y el seguimiento de actividad se realizará mediante el método de enol-borato, dopacromo y Ellman para HPPD, tirosinasa y AChE,respectivamente. Posterior a determinar el nivel de actividad (IC50) de cada compuesto aislado se estudiará el posible efecto sinergista que pudieran ejercer al combinarlos entre ellos (si más de un compuesto es aislado de una planta) y con compuestos comerciales. Si los resultados muestran que los extractos ensayados y los metabolitos activos presentes en ellos exhiben alta efectividad en inhibir las enzimas, ellos pueden surgir como agentes terapéuticos eficaces para el tratamiento de ciertas enfermedades que las involucran y de esta manera mejorar la calidad de vida de los pacientes afectados. Estos productos pueden dar lugar a las compañías farmaceúticas a producir drogas no convencionales como nuevas alternativas medicinales. Por otro lado estas sustancias pueden derivar en novedosos herbicidas, antimicrobianos o insecticidas.Dado que todas las plantas propuestas crecen fácilmente en Argentina, la producción de estos medicamentos significaría nuevas fuentes laborales para nuestro país. El hecho de poder obtener y posteriormente utilizar estos productos aumenta en forma sustancial el aprovechamiento que podemos darle a nuestra rica flora nativa.

Los objetos técnicos y los organismos tecnificados. Reflexiones filosóficas, estéticas y políticas sobre la técnica. Technical objects and technified organisms. Philosophical, aesthetical and political approaches on technique

El presente proyecto se propone como parte inicial de una investigación sobre la relación entre naturaleza/cultura/técnica. Tradicionalmente la naturaleza y la cultura se han considerado como ámbitos diferenciados y opuestos. Y es en esta distinción donde la técnica adquiere un lugar central. El pensamiento occidental sobre la técnica ha recibido diversas interpretaciones: desde una subordinación con respecto al conocimiento verdadero (episteme) en la filosofía clásica, un optimismo sobre la técnica como posibilidad de dominación de la naturaleza en el Renacimiento y la Ilustración, y la ambigüedad y desasosiego romántico (Mitcham, 1979). Durante el siglo XX se distinguen dos posiciones antagónicas sobre la técnica. Por un lado, una actitud “crítica” donde pueden identificarse los trabajos de filósofs de diferentes tradiciones como Ortega y Gasset (1939), Heidegger (1954), Mumford (1971) Ellul (1960) y la Escuela de Frankfurt. Por otro lado, una filosofía de la técnica “ingenieril” que consiste en el análisis de la tecnología como un paradigma de pensamiento y acción humana. Esta dicotomía ha sido interpretada por Eco como “apocalípticos e integrados”. Más allá de las mencionadas diferencias, lo que tienen en común ambas posiciones es que parten de una dicotomía entre cultura y naturaleza. Nuestra perspectiva rechaza esta dicotomía, por el contrario, evidenciamos una creciente imbricación entre ambas donde las fronteras entre una y otra se hacen difusas. La noción de “objeto técnico” propuesta por Simondon (2007) hace referencia a la inserción del objeto técnico en la cultura, donde debe reconocerse la “realidad humana” presente en el mismo. Ahora bien, esto no significa “humanizar el objeto técnico”, sino más bien indagar sobre el lugar que este ocupa en la cultura como también establecer su relación con la naturaleza. En el siglo XVII el hombre mismo es reinterpretado como máquina (La Mettrie, 2000). En la actualidad pueden identificarse dos tendencias en la concepción de la técnica: los «humanos-máquinas» y las «máquinas-humanas», en otras palabras, la disposición del humano hacia la máquina y la tendencia de la máquina hacia lo humano. No obstante, ambas posiciones siguen manteniendo una distinción taxonómica entre el cuerpo –o lo orgánico- y lo maquínico, lo que implica una consideración de esta relación de manera extrínseca. Frente a esta tensión Haraway propone el concepto de cyborg: «un organismo cibernético» (1995). Los desarrollos tecnológicos han producido una modificación tal en la vida de los seres orgánicos en los cuales ya no puede concebirse su cuerpo independientemente de la tecnología. Esto conduce a replantear la distinción entre “animales/hombres/máquinas”, entendiendo a los mismos como expresiones de naturaleza, cultura y tecnología respectivamente. Nuestra investigación parte de la hipótesis que la técnica diluye diferencias de orden natural y cultural a través de los objetos técnicos que son productos culturales. La estética se ocupa de la percepción sensible del mundo no puede eludir su dimensión técnica. Al margen de la crítica a la “Industria cultural” consideramos relevante la aproximación de Benjamin al problema de la técnica porque aborda la imbricación antes mencionada en el campo de la percepción. Según Benjamin la irrupción de la técnica al mismo tiempo que posibilita una estetización de la política que confluye en el fascismo como punto extremo también abre la posibilidad de desmontar la ideología del progreso infinito (1967). Una integración entre aproximaciones estéticas y políticas a la técnica Flusser (1983) propone la “caja negra” como metáfora de la técnica contemporánea. Su propuesta es la “apertura de la caja negra” que consiste en tomar conocimiento del funcionamiento del dispositivo. Nuestra propuesta de investigación aborda la técnica desde una consideración filosófica/estética/política, donde redefiniremos la técnica partiendo de la imbricación entre cultura y naturaleza. This project will set the basis for a sustained research on the relation nature/culture/technique. They have been traditionally considered as separate and even opposite fields. And it is on the brink of this distinction where technique plays a central role. In Western thought technique has received many interpretations since the beginnings of philosophy: from a subordination to true knowledge (episteme) in classic philosophy, or the optimism which sees in technique the possibility of dominating nature in the Renaissance and in the Enlightenment, to the Romantic ambiguity and uneasiness towards technological change (Mitcham, 1979). During the twentieth century two opposed approach on technique prevail. On one hand, a “critical” attitude such defines the work of philosophers of different traditions such as Ortega y Gasset (1939), Heidegger (1954), Mumford (1971) Ellul (1960) and the Frankfurt School. On the other hand there is an “engineering” philosophy of technique that consists in the analisis of technology as a paradigm to understand human action and thought. Besides their differences, both positions have in common a dichotomy between nature and culture. We reject such dichotomy. On the contrary we consider there is a growing intertwinement between both which blurs the borders of the concepts. Simondon’s notion of “technical object” refers to the insertion of the technique in culture where the “human reality” in it must be recognised. This does not imply “humanising the technical object”, but investigate on the role it plays on culture and establishing its relation to nature. To articulate this relation we will work with unorthodox approaches on technique such as Benjamin (1967), Flusser (1983) and others. The hypothesis of our project is that the traditional distinction of “animal/man/machine” must be re-thought, therefore raising the question on the blurring line between nature, culture and technique and its effects in philosophy, politics and aesthetics.