Solid Phase Microextraction as a Sample Preparation Tool for Targeted and Untargeted Analysis of Biological Matrices

Successful determination of small molecules in complex biological systems requires implementation of robust analytical methodologies able to provide reliable information in a cost-effective and efficient manner. Solid phase microextraction (SPME) is a versatile, non-exhaustive sample preparation tool that has been demonstrated to be well-suited for facile analysis of biological matrices such as plasma, blood, and tissue. In SPME, a small amount of extraction phase immobilized on a solid support is utilized for the extraction of analytes of interest, either from the sample headspace or by direct immersion of the fiber in the matrix of choice. For the analysis of non-volatile compounds in complex biological matrices, SPME coatings made of sorbents embedded in a biocompatible binder (e.g. polyacrylonitrile (PAN)) are directly immersed into the sample under study for a defined period of time so as to allow for sufficient and reproducible extraction of analytes. The main advantages of such coating materials rely on their ability to provide high selectivity for extraction of small molecules; their aptness for immobilization in different support geometries; their inertness and robustness, which even enables their reusability in complex biological matrices; and their suitability towards in vivo extractions. In view of these advantages, the body of this doctoral thesis presents novel applications and developments of SPME for both targeted and untargeted analysis of different biological matrices such as biofluids and brain tissue. The first part of the research conducted for this thesis encompasses the application of SPME in thin-film format for high throughput determination of multiple prohibited substances in plasma. A biocompatible SPME extraction phase made of hydrophilic-lipophilic balance (HLB) particles immobilized with PAN was employed for extractions, demonstrating satisfactory extraction capabilities for 25 compounds of a wide range of polarities (logP from -2 to 6.8). By taking full advantage of the 96 thin-film handling capability of the automated system, a preparation time of approximately 1.5 min per sample can be achieved. Rewarding results in terms of absolute matrix effects were found for the majority of the studied analytes, given that 24 out of 25 compounds exhibited values in the range 100 - 120%. The method was validated in terms of linearity (R2> 0.99), inter and intra-day accuracy (85 – 130%) and precision (< 20%), and limits of quantitation (0.25 – 10 ng mL-1 for most compounds). Based on the positive results obtained after employing the developed method for the analysis of doping compounds in plasma samples, and considering the need for cost-effective and single use devices, the possibility of employing alternative materials to manufacture SPME devices was explored. To that end, new thin-film SPME devices prepared on plastic as potential single-use samplers for bioanalysis were developed and tested. Polybutylene terephthalate (PBT) was selected as a support based on its chemical resistance, low cost, and suitability as a material for different medical grade components. The proposed devices were assessed in terms of robustness, chemical stability, and possible interferences upon exposure to different solvents and matrices. Satisfactory results were obtained upon utilization of the manufactured samplers for the quantitation of multiple drugs in biofluids such as urine, plasma, and whole blood. Interestingly, our results showed that more than 20 extractions in complex biofluids can be performed without incurring significant changes in coating performance. These findings evidenced the robustness of PAN-based coatings applied on polymeric substrates, and opened up opportunities for the introduction of new support materials for manufacture of SPME biocompatible devices aimed at a wide range of applications. Taking into account that SPME is a non-exhaustive extraction technique where analytes are extracted via free concentration, assessing the effect of variable matrix composition on final SPME recoveries is invaluable in avoiding biased results. With this in mind, part of this thesis also involved the investigation of the effect of hematocrit (Hct) levels on SPME extractions from whole blood. The obtained results demonstrated that the Hct effect in SPME is dependent on the analytes of interest, and that different outcomes can be attained by varying experimental conditions such as coating type, convection, and extraction time. Interestingly, the relative affinities of target compounds for matrix components and coating materials were demonstrated to be one of the main determining factors on the final effect that erythrocyte levels impart on SPME recoveries. Although Hct content was shown to affect the extraction of each analyte differently, and be dependent on experimental parameters, correction of matrix variability is enabled through the use of appropriate internal standards. In view of the rewarding results obtained in the analysis of a broad range of target analytes, SPME in its fibre configuration was evaluated based on its performance for untargeted analysis of brain tissue. For that purpose, the metabolite coverage provided by C18, mixed mode (MM), and HLB 7 mm fibres following extraction from brain homogenate at static conditions was assessed. Our results demonstrated that for compounds of medium to high polarity, both HLB and MM coatings were able to offer similar coverage at the same desorption conditions. For extraction of lipids, C18 and HLB exhibited the best recoveries with the use of 1:1 methanol:isopropyl alcohol as desorption solvent. Interestingly, the use of different desorption solvents was found to greatly influence the final composition of the brain extract obtained via SPME. Other parameters such as extraction time, coating washing step, and inter-fibre reproducibility were also considered and discussed. Lastly, the balanced metabolite coverage provided by SPME was successfully utilized for in vivo monitoring of metabolic changes occurring in the hippocampus of rat brain after electrical stimulation (DBS) of the ventromedial prefrontal cortex (vmPFC), which has been previously shown to induce anti-depressant like effects in rodents. The use of in vivo SPME enabled the monitoring of significant variations, not only among small polar metabolites such as amino acids, but also in lipids belonging to different classes. Compounds such as citrulline, glutamate, taurine, uric acid, sphingomyelins, and phosphatidylethanolamines, among others, exhibited statistically significant changes after acute exposure of animals to electrical stimulation for 3 hours. Although additional studies are needed to establish the contribution of the biochemical changes observed in this study to the effect of DBS in the treatment of depression, our work provided new directions towards a better understanding of the mechanisms taking place in the brain upon application of electrical stimulation to the vmPFC

Evaluation and Application of Microextraction Techniques Coupled with Portable Analytical Instrumentation for On-site Analysis

In recent years, on-site analysis has garnered increased interest from the scientific community. The development of smaller, more sophisticated analytical instruments, and the establishment of new environmental regulations have encouraged the application of new methodologies for field analysis. Prominent advantages of on-site analysis include elimination of error sources due to sample transportation and matrix modification, considerable reduction in analysis time, and more accurate and precise analytical results. Several techniques suitable for on-site analysis, which integrate sampling and sample preparation in one step, have demonstrated high versatility and throughput in field applications. This research was focused on the application and evaluation of three microextraction techniques: solid phase microextraction (SPME), needle trap devices (NTD) and membrane extraction with sorbent interface (MESI), which were then coupled with various portable instruments for on-site analysis of different systems. Additionally, the conducted project involved the development of an approach using ion mobility spectrometry detection (IMS) coupled with a miniaturized gas chromatograph (GC) as a powerful system for field analysis. This proposed GC-IMS exhibited satisfactory performance in terms of retention time (inter-day variation < 3%) and response stability (intra and inter-day relative standard deviations (RSDs) < 10 %). Moreover, when coupled with NTD, it showed limits of detection comparable to those provided by conventional benchtop instruments. Other portable GC instruments employed in this project included flame ionization and mass spectrometry detection. Three different sample systems were investigated using SPME and NTD together with these portable instruments: emissions of a pine branch, breath samples, and indoor pollutants in a polymer synthesis laboratory. Consequently, the feasibility of using SPME and NTD for determination of free and total concentrations was investigated. Finally, MESI was successfully coupled with the newly proposed GC-IMS system, and its functionality was evaluated by analyzing acetone in breath samples

Presence of matrix vesicles in the body of odontoblasts and in the inner third of dentinal tissue: a scanning electron microscopyc study

Objectives: The aim of this report is to present the results of a scanning electron microscopic study on the presence of matrix vesicles (MVs) found in human dentine. Study Design: Dentin tissue from 20 human bicuspids was analyzed by means of scanning electron microscopy. Results: MVs were found as outgrowths of the cellular membrane of the odontoblastic body, the more proximal portion of the odontoblastic process before entering the dentinal tubule and in the odontoblastic process within the inner third of the dentin. Size of MVs varied depending on location. In the inner third of dentin, they were seen in diverse positions; as membranal outgrowths, deriving from the odontoblastic process, lying free in the intratubular space and attached to the dentinal wall. Sometimes, they were seen organized forming groups of different sizes and shapes or as multivesicular chains running from the surface of the odontoblastic process to the tubular wall. MVs were present in places never considered: 1) the body of odontoblasts; 2) the most proximal part of the odontoblastic processes before entering the circumpulpal dentine and also: 3) in the inner third of dentinal tissue. Conclusions: According to our results, MVs not only participate during mantle dentin mineralization during early dentinogenesis, they also contribute during the mineralization process of the inner dentin

Las reparaciones en espacios transicionales y sus perplejidades. La aparente transformación del derecho de daños

126 páginasEn el escenario de justicia transicional colombiano los daños causados por terceros excombatientes o por los servidores públicos con dolo o culpa grave en el conflicto armado son garantizados por el Estado. Al respecto surgen distintas preguntas ¿cuál es la justificación iusfilosófica para esta garantía del deber de reparar? ¿Cómo le es imputable causal y jurídicamente estos comportamientos al Estado? ¿Merece el Estado ser condenado por tales eventos? Dichas preguntas son abordadas en clave teórico-filosófica en el artículo que sigue. Para ello, se utilizan teorías modernas sobre el rol del derecho de daños, como distribución de derechos y deberes de indemnidad, y las exigencias a las que responde, tanto en escenarios de normalidad como en escenarios de transición desde el concepto de planes sociales esbozado por Shapiro en Legalidad. Con base en este sustento epistemológico se analiza el Acuerdo de Paz de La Habana, así como las normas que le desarrollan, para concluir, que la justicia transicional permite moldear las instituciones básicas de la responsabilidad con una finalidad social: la superación del mal causado, la redención de los responsables, el revestimiento de las víctimas y el fortalecimiento del Estado social de Derecho.The scenario of Colombian transitional justice implies that damages caused by ex-combatant third parties or by public servants with intent or serious fault in the armed conflict are guaranteed by the State. In this regard, different questions arise: what is the philosophical justification for this guarantee of the duty to repair? How are these behaviors legally and causally attributable to the State? Does the State deserve to be convicted of such events? In this article, these questions are addressed in a theoreticalphilosophical key. For this, modern theories are used on the role of the Tort law, such as the distribution of rights and obligations of indemnity, and the demands to which it responds, both in normality scenarios and in transition scenarios from the concept of social plans outlined by Shapiro in Legality. Based on this epistemological sustenance, the Havana Peace Agreement is analyzed, as well as the norms that develop it, to conclude, that transitional justice allows shaping the basic institutions of responsibility with a social purpose: overcoming the evil caused, the redemption of those responsible, the lining of the victims and the strengthening of the social rule of law.Magíster en Justicia y Tutela de los Derechos con énfasis en Teoría Jurídica y Filosofía del DerechoMaestrí

Cómo algunas barreras y cargas afectan la actividad emprendedora motivada por oportunidad y necesidad

Entrepreneurship has been regarded as an important input for the economic growth. It does not only fetch employment, which entails development, but also fosters innovation and productivity. Nevertheless, Entrepreneurs face many social, economic and political barriers and burdens. The aim of this paper is to find if there is a significant effect of these barriers and burdens on opportunity and necessity driven entrepreneurial activity in Latin American and the Caribbean countries. A sample of the available data for 12 countries was selected from the Global Entrepreneurship Monitor (GEM) database and Doing Business annual report. To evaluate the hypotheses, a panel data econometric model was used. As a result, there is a stronger negative impact on opportunity-driven than on necessity-driven entrepreneurship. The paper ends with some advice on policy decisions that could foster entrepreneurial activity in the region.El emprendimiento es considerado como insumo importante para el crecimiento económico, ya que a más de generar empleo, fomenta la innovación y la productividad. Sin embargo, los emprendedores enfrentan barreras y cargas sociales, económicas y políticas. El objetivo de este artículo es determinar si existe un efecto significativo de estas barreras y cargas en la actividad empresarial impulsada por las oportunidades y la necesidad en los países de América Latina y el Caribe. Para evaluar las hipótesis se seleccionó una muestra de los datos disponibles para 12 países de la base de datos Global Entrepreneurship Monitor (GEM) y el informe anual de Doing Business, y se elaboró un modelo econométrico de datos de panel. Como resultado, hay un impacto negativo más fuerte en el emprendimiento impulsado por las oportunidades que por la necesidad. Al final se aconseja sobre decisiones políticas que podrían fomentar la actividad empresarial en la región

Propuesta de programa de preparación a la jubilación. La jubilación como transición ocupacional.

Los cambios demográfico-económicos que se están produciendo en las últimas décadas han llevado a plantear la viabilidad del Estado de Bienestar y el Sistema de Pensiones. Esto, supone un riesgo para la calidad de vida de la persona mayor (a corto y a largo plazo). Para minimizar efectos negativos, se proponen los programas de preparación a la jubilación como herramienta preventiva de enfoque biopsicosocial. Desde terapia ocupacional se analizan los riesgos de la jubilación entendida como transición ocupacional y se ofrecen estrategias para una adecuada adaptación. El programa, que consta de tres fases (evaluación de los usuarios, capacitación y evaluación del programa), va dirigido a aquellas personas que se encuentran en el año previo a la jubilación y sus parejas. El objetivo que se plantea desde terapia ocupacional es: mejorar la calidad de vida de la persona mayor mediante una adecuada transición a la jubilación basada en el empoderamiento y la justicia ocupacional. Los marcos y modelos que se han utilizado para el desarrollo de este programa son: M.O.H, C.M.O.P y el Marco de trabajo. Las conclusiones se recogen al final

Plan de Empresa: Almazara Reina del Ebro

Este estudio pretende elaborar un plan de empresa para la creación de una almazara. Analizaremos los factores externos e internos, el sector oleícola español y el mercado del aceite de oliva, para tomar conciencia de la situación actual del mercado. Plantearemos como va a ser la empresa desde la que vamos a operar, explicando desde la normativa jurídica y legal, hasta el producto que vamos a vender y su proceso de fabricación, elaboraremos un plan de marketing con el cual definiremos la estrategia de distribución y comunicación. Estableceremos una estrategia de precios y definiremos la estrategia de ventas. Finalmente con los datos analizados vamos a preparar un estudio económico para comprobar que el proyecto es viable y haremos una previsión de la rentabilidad del mismo según diferentes escenarios

Simplified colorless characterization scheme for coherent receivers in DWDM scenarios using a single interferer

Intradyne coherent receivers, capable of detecting an individual wavelength-division multiplexed channel just by tuning the local oscillator frequency, is of great interest for the development of high-capacity flexible optical networks. Nevertheless, the unavoidable amplitude imbalances inherent to any realistic coherent receiver induce an interference contribution from the self-beating of the coincident channels present at its input. The charac- terization of this degraded colorless reception operation is of fundamental importance, but it usually requires the use of rather complex experimental setups, especially when the effects of tens of interference channels should be evaluated. In this work we propose a novel experimental setup that only requires the use of a single intense interferer to emulate those coincident channels, thus drastically simplifying the characterization process. In addition, we develop a general expression for the signal-to-noise ratio of the system that theoretically justifies the intended setup and demonstrate by massive numerical simulations its accuracy in different scenarios. We believe that the proposed approach may contribute to facilitate the experimental characterization of high-performance colorless coherent receiversFunding for open access charge: Universidad de Málaga / CBU

Análisis de los Determinantes y Costos de la Informalidad en las Pequeñas Empresas: Un Estudio Empírico para el Perú // Analysis of Determinants and Costs of Informality in Small Businesses: An Empirical Study for Peru

Informality in small businesses is a widespread phenomenon in Latin American countries. In Peru it represents 50% of the business units, a situation that implies the knowledge of the determinants of this reality to contribute to the design of public policies. Thus, the present work studies the variables related to the productive performance and the life quality of the company owner’s home as elements highly related to informality. In addition, it is evaluated whether the employer’s decision about informality responds to a maximization of profits or, on the contrary, it is due to exclusion because the current legislation is apart from the real nature of the business unit. The conclusions allow observing that the characteristics of the home facilitate the formality and that the existing tax regulatory framework excludes small companies, which experience lower levels of profit compared to formal companies with similar characteristics.La informalidad en la pequeña empresa es un fenómeno ampliamente extendido en los países de Latinoamérica. En el Perú representa el 50% de las unidades empresariales, situación que supone conocer cuáles son los determinantes de esta realidad para contribuir al diseño de políticas públicas. Es así que la presente investigación estudia las variables relacionadas con el desempeño productivo y la calidad de vida del hogar del dueño de la empresa como elementos altamente vinculados a la informalidad. Además, se evalúa si la decisión del empresario sobre la informalidad responde a una maximización de beneficios o, por el contrario, se debe a una exclusión debido a que la legislación actual es ajena a la real naturaleza de la unidad de negocio. Las conclusiones permiten observar que las características del hogar facilitan la formalidad y que el marco normativo tributario existente excluye a las pequeñas empresas, que experimentan menores niveles de ganancia en comparación con empresas formales de características similares. ------------------------------------ Informality in small businesses is a widespread phenomenon in Latin American countries. In Peru it represents 50% of the business units, a situation that implies the knowledge of the determinants of this reality to contribute to the design of public policies. Thus, the present work studies the variables related to the productive performance and the life quality of the company owner’s home as elements highly related to informality. In addition, it is evaluated whether the employer’s decision about informality responds to a maximization of profits or, on the contrary, it is due to exclusion because the current legislation is apart from the real nature of the business unit. The conclusions allow observing that the characteristics of the home facilitate the formality and that the existing tax regulatory framework excludes small companies, which experience lower levels of profit compared to formal companies with similar characteristics