No description available.

<p>The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. </p><p>In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. </p><p>The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.</p>

<p>Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed.</p><p>Microdialysis was used <i>in vitro</i> for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected <i>in vivo,</i> was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues.</p><p>Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis. </p>

The analysis of low abundant biological molecules is often challenging due to their chemical properties, low concentration and limited sample volumes. Neuropeptides are one group of molecules that fits these criteria. Neuropeptides also play an important role in biological functions, which makes them extra interesting to analyze. A classic chemical analysis involves sampling, sample preparation, separation and detection. In this thesis, an enhanced solid supported microdialysis method was developed and used as a combined sampling- and preparation technique. In general, significantly increased extraction efficiency was obtained for all studied peptides. To be able to control the small sample volumes and to minimize the loss of neuropeptides because of unwanted adsorption onto surfaces, the subsequent analysis steps were miniaturized to a micro total analysis system (µ-TAS), which allowed sample pre-treatment, injection, separation, manipulation and detection. In order to incorporate these analysis functions to a microchip, a novel microfabrication protocol was developed. This method facilitated three-dimensional structures to be fabricated without the need of clean room facilities. The sample pre-treatment step was carried out by solid phase extraction from beads packed in the microchip. Femtomole levels of neuropeptides were detected from samples possessing the same properties as microdialysates. The developed injection system made it possible to conduct injections from a liquid chromatographic separation into a capillary electrophoresis channel, which facilitated for advanced multidimensional separations. An electrochemical sample manipulation system was also developed. In the last part, different electrospray emitter tip designs made directly from the edge of the microchip substrate were developed and evaluated. The emitters were proven to be comparable with conventional, capillary based emitters in stability, durability and dynamic flow range. Although additional developments remain, the analysis steps described in this thesis open a door to an integrated, on-line µ-TAS for neuropeptides analysis in complex biological samples.

Advances in life science require knowledge of active molecules in complex biological systems. These molecules are often only present for a certain time and at limited concentrations. Integrated micro-analytical tools for sampling, separation and mass spectrometric (MS) detection would meet these requests and are therefore continuously gaining interest. An on-line coupling of analytical functions provides shorter analysis time and less manual sample handling. In this thesis, improved compatibility of microdialysis sampling and multidimensional separations coupled to MS detection are developed and discussed. Microdialysis was used in vitro for determination of the non-protein bound fraction of the drug ropivacaine. The sampling unit was coupled on-line to capillary column liquid chromatography (LC) followed by ultraviolet or MS detection. For MS detection, the system was extended with a desalting step and an addition of internal standard. A method for MS screening of microdialysates, collected in vivo, was also developed. The method involved sampling and measurements of the chemical pattern of molecules that generally are ignored in clinical investigations. Chemometric tools were used to extract the relevant information and to compare samples from stimulated and control tissues. Complex samples often require separation in more than one dimension. On-line interfaces for sample transfer between LC and capillary electrophoresis (CE) were developed in soft poly(dimethylsiloxane) (PDMS). MS detection in the LC-CE system was optimised on frequent sampling of the CE peak or on high resolution in mass spectra using time-of-flight (TOF)MS or Fourier transform ion cyclotron resonance (FTICR)MS, respectively. Aspects on electrode positioning in the LC-CE interface led to development of an on-column CE electrode. A successful method for deactivation of the PDMS surface using a polyamine polymer was also developed. The systems were evaluated using peptides and proteins, molecules that are gaining increased attention in bioscience, and consequently also in chemical analysis.

No description available.

O Paradigma Orientado a Objetos tem sido atualmente a abordagem dominante de desenvolvimento de software. Contudo, ela sofre da Tirania da Decomposição Dominante, pois não permite uma modularização adequada da implementação relativa a interesses estruturais. Como consequência, a implementação relativa a cada interesse estrutural fica espalhada pelos módulos do programa e entrelaçada com a implementação relativa a outros interesses estruturais. Outras abordagens de desenvolvimento de software, como o Desenvolvimento de Software Orientado a Aspectos com AspectJ e a Separação Multidimensional de Interesses em Hiperespaços com Hyper/J e CME, atingem sucesso moderado em oferecer mecanismos que permitem superar as deficiências do Paradigma Orientado a Objetos. No entanto, tais abordagens também possuem deficiências e omissões que devem ser reparadas para que elas possam se tornar utilizáveis em contextos típicos de desenvolvimento de software complexo. Este trabalho especifica uma nova abordagem, denominada Desenvolvimento de Software Orientado a Temas (DSOT), que tem como objetivo superar algumas deficiências das abordagens anteriores por meio de mecanismos que permitem a manipulação da implementação de cada interesse estrutural de forma separada e a manipulação da implementação de cada tipo de dado de forma separada. Além disso, DSOT possui operadores que são ortogonais, isto é, podem ser utilizados de forma combinada ou separada, para efetuar a composição de módulos do programa. Mostra-se o modelo conceitual do DSOT e descrevese um estudo de caso que consiste no desenvolvimento de um programa para demonstrar mais concretamente como o DSOT funciona na prática. Não se demonstra a superioridade do DSOT para o caso geral, mas os resultados alcançados evidenciam que o DSOT é uma abordagem promissora que merece ser investigada mais aprofundadamente em pesquisas futuras / The Object-Oriented Paradigm has currently been the dominant approach for developing software. However, it suffers from the Tyranny of the Dominant Decomposition, as it does not support a suitable modularization to the implementation relative to structural concerns. As a consequence, the implementation relative to each structural concern is scattered throughout the program modules and tangled with the implementation relative to other structural concerns. Some software development approaches, such as Aspect-Oriented Software Development with Aspect and Multidimensional Separation of Concerns in Hyperspaces with Hyper/J and CME, achieve moderate success in offering mechanisms that make it possible to overcome the deficiencies of the Object-Oriented Paradigm. However, such approaches also possess deficiencies and ommissions that must be corrected in order for them to get usable in typical complex software development contexts. This work specifies a new approach, named Theme- Oriented Software Development (TOSD), which aims at overcoming some deficiencies from previous approaches through mechanisms that support the handling of implementation for every structural concern separately and the handling of implementation for every data type separately. Moreover, TOSD contains operators which are orthogonal, that is, they can be used separately or as a combination, in order to perform composition of the program modules. We show the conceptual model of TOSD and describe a case study which consists in the development of a program to demonstrate more concretely how TOSD works in practice. We do not demonstrate the superiority of TOSD for the general case, but the results we have obtained suggest that TOSD is a promissing approach which deserves a deeper investigation in future research

O Paradigma Orientado a Objetos tem sido atualmente a abordagem dominante de desenvolvimento de software. Contudo, ela sofre da Tirania da Decomposição Dominante, pois não permite uma modularização adequada da implementação relativa a interesses estruturais. Como consequência, a implementação relativa a cada interesse estrutural fica espalhada pelos módulos do programa e entrelaçada com a implementação relativa a outros interesses estruturais. Outras abordagens de desenvolvimento de software, como o Desenvolvimento de Software Orientado a Aspectos com AspectJ e a Separação Multidimensional de Interesses em Hiperespaços com Hyper/J e CME, atingem sucesso moderado em oferecer mecanismos que permitem superar as deficiências do Paradigma Orientado a Objetos. No entanto, tais abordagens também possuem deficiências e omissões que devem ser reparadas para que elas possam se tornar utilizáveis em contextos típicos de desenvolvimento de software complexo. Este trabalho especifica uma nova abordagem, denominada Desenvolvimento de Software Orientado a Temas (DSOT), que tem como objetivo superar algumas deficiências das abordagens anteriores por meio de mecanismos que permitem a manipulação da implementação de cada interesse estrutural de forma separada e a manipulação da implementação de cada tipo de dado de forma separada. Além disso, DSOT possui operadores que são ortogonais, isto é, podem ser utilizados de forma combinada ou separada, para efetuar a composição de módulos do programa. Mostra-se o modelo conceitual do DSOT e descrevese um estudo de caso que consiste no desenvolvimento de um programa para demonstrar mais concretamente como o DSOT funciona na prática. Não se demonstra a superioridade do DSOT para o caso geral, mas os resultados alcançados evidenciam que o DSOT é uma abordagem promissora que merece ser investigada mais aprofundadamente em pesquisas futuras / The Object-Oriented Paradigm has currently been the dominant approach for developing software. However, it suffers from the Tyranny of the Dominant Decomposition, as it does not support a suitable modularization to the implementation relative to structural concerns. As a consequence, the implementation relative to each structural concern is scattered throughout the program modules and tangled with the implementation relative to other structural concerns. Some software development approaches, such as Aspect-Oriented Software Development with Aspect and Multidimensional Separation of Concerns in Hyperspaces with Hyper/J and CME, achieve moderate success in offering mechanisms that make it possible to overcome the deficiencies of the Object-Oriented Paradigm. However, such approaches also possess deficiencies and ommissions that must be corrected in order for them to get usable in typical complex software development contexts. This work specifies a new approach, named Theme- Oriented Software Development (TOSD), which aims at overcoming some deficiencies from previous approaches through mechanisms that support the handling of implementation for every structural concern separately and the handling of implementation for every data type separately. Moreover, TOSD contains operators which are orthogonal, that is, they can be used separately or as a combination, in order to perform composition of the program modules. We show the conceptual model of TOSD and describe a case study which consists in the development of a program to demonstrate more concretely how TOSD works in practice. We do not demonstrate the superiority of TOSD for the general case, but the results we have obtained suggest that TOSD is a promissing approach which deserves a deeper investigation in future research