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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Micropatterning and microelectrochemical characterisation of biological recognition elements

Turcu, Eugen Florin. January 2004 (has links) (PDF)
Bochum, University, Diss., 2004.
2

Automatisches Segmentieren von Mikroarraybildern

Katzer, Mathias. January 2004 (has links) (PDF)
Bielefeld, Universiẗat, Diss., 2004.
3

Entwicklung eines fluoreszenz-optischen Evaneszenzfeldmesssystems für die Echtzeitanalyse von DNA-Mikroarrays

Lehr, Hans-Peter. January 2002 (has links) (PDF)
Freiburg (Breisgau), Universiẗat, Diss., 2002.
4

Diagnosis of human sub-telomeric chromosomal deletions by Microarray

Darmanian, Artur Pavlovich, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2008 (has links)
ABSTRACT A major cause of genetic disease is associated with chromosomal imbalances, such as deletions (subtelomeric, terminal and interstitial), duplications, and unbalanced translocations present in a particular chromosome segment. The diagnosis of many genetic diseases remains problematic. This is due in part to difficulty in detection of DNA copy number changes, when these are either too small (for conventional cytogenetics) or too large, for standard molecular approaches. From this viewpoint, the development of new screening methods with improvement of resolution is very important. Genome-wide screening at a molecular level began to appear feasible with the completion of the human genome sequence. From this beginning, high-resolution whole-genome technologies could be envisaged, to improve the diagnostic detection rate for even the smallest of chromosomal imbalances. The technique known as ???array-based comparative genomic hybridization??? (array-CGH) does allow such a high-resolution screening, by use of reference DNA probes, printed onto arrays, thus consisting of thousands of genomic clones. In this study we extensively investigated many major aspects of array-CGH technology from preparation of microarray probes and printing microarray slides, to a development of custom protocols and custom softwares for data processing and analysis. We have trailed several array types and protocols, direct and indirect DNA labelling techniques and, as a result, we have achieved the practical application which was our target at the onset of this work. This was to use a modified array-CGH method, as a robust and economical diagnostic test in detection of deletions and duplications within the human genome. The project has been successful, in terms of one very important outcome: The laboratory in which this work was done is now the leading clinical diagnostic lab in this field, in Australia???s most populous state of New South Wales. That achievement would not have been possible without a very lengthy period of developmental work, including that which comprises much of this thesis.
5

Diagnosis of human sub-telomeric chromosomal deletions by Microarray

Darmanian, Artur Pavlovich, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW January 2008 (has links)
ABSTRACT A major cause of genetic disease is associated with chromosomal imbalances, such as deletions (subtelomeric, terminal and interstitial), duplications, and unbalanced translocations present in a particular chromosome segment. The diagnosis of many genetic diseases remains problematic. This is due in part to difficulty in detection of DNA copy number changes, when these are either too small (for conventional cytogenetics) or too large, for standard molecular approaches. From this viewpoint, the development of new screening methods with improvement of resolution is very important. Genome-wide screening at a molecular level began to appear feasible with the completion of the human genome sequence. From this beginning, high-resolution whole-genome technologies could be envisaged, to improve the diagnostic detection rate for even the smallest of chromosomal imbalances. The technique known as ???array-based comparative genomic hybridization??? (array-CGH) does allow such a high-resolution screening, by use of reference DNA probes, printed onto arrays, thus consisting of thousands of genomic clones. In this study we extensively investigated many major aspects of array-CGH technology from preparation of microarray probes and printing microarray slides, to a development of custom protocols and custom softwares for data processing and analysis. We have trailed several array types and protocols, direct and indirect DNA labelling techniques and, as a result, we have achieved the practical application which was our target at the onset of this work. This was to use a modified array-CGH method, as a robust and economical diagnostic test in detection of deletions and duplications within the human genome. The project has been successful, in terms of one very important outcome: The laboratory in which this work was done is now the leading clinical diagnostic lab in this field, in Australia???s most populous state of New South Wales. That achievement would not have been possible without a very lengthy period of developmental work, including that which comprises much of this thesis.
6

Detecting Locus-Locus Interactions Using Microarray Data

Gao, YanFei 04 1900 (has links)
In this report we explore how to find the locus-locus interaction using microarray data. Our analysis makes use of a dataset from an experiment with Affymetrix GeneChip MGU74Av2 for mice. In Chapter 1 we give the genetics background, an introduction to microarray methodology and the preprocessing of microarray data, and a review of SAM (Significance Analysis of Microarrays) method for finding differentially expressed genes in microarray data. In Chapter 2 we describe our dataset and our objective of finding the genes with locus-locus interaction but with no main effect. We also show how to find the interaction in this chapter. In Chapter 3 we show the simulation study of detecting the locus-locus interaction without main effects and propose a two-stage method of doing that. In Chapter 4 we apply the two-stage method to the microarray data and focus on the second stage analysis. In Chapter 5 we examine an alternative method using bootstrap resampling in place of permutations. Chapter 6 contains our conclusion and some suggestions for future research. / Thesis / Master of Science (MS)
7

Advances in protein microarray technology for glycomic analysis

Propheter, Daniel Champlin 13 October 2011 (has links)
The cell surface is enveloped with a myriad of carbohydrates that form complex matrices of oligosaccharides. Carbohydrate recognition plays crucial and varying roles in cellular trafficking, differentiation, and bacterial pathogenesis. Lectin microarray technology presents a unique platform for the high-throughput analysis of these structurally diverse classes of biopolymers. One significant hinderance of this technology has been the limitation imposed by the set of commercially available plant lectins used in the array. To enhance the reproducibility and scope of the lectin panel, our lab generated a small set of bacteria-derived recombinant lectins. This dissertation describes the unique advantages that recombinant lectins have over traditional plant-derived lectins. The recombinant lectins are expressed with a common fusion tag, glutathione-S-transferase (GST), which can be used as an immobilization handle on glutathione (GSH)-modified substrates. Although protein immobilization via fusion tags in a microarray format is not novel, our work demonstrates that protein activity through site-specific immobilization is enhanced when the protein is properly oriented. Although orientation enhanced the activity of our GST-tagged recombinant lectins, the GSH-surface modification precluded the printing of non-GST-tagged lectins, such as the traditional plant lectins, thus limiting the structural resolution of our arrays. To solve this issue, we developed a novel print technique which allows the one-step deposition and orientation of GST-tagged proteins in a microarray format. To expand our view of the glycome, we further adapt this method for the in situ orientation of unmodified IgG and IgM antibodies using GST-tagged antibody-binding proteins. Another advantage of recombinant lectins is in the ease of genomic manipulation, wherein we could tailor the binding domain to bind a different antigen. We demonstrate this by producing non-binding variants of the recombinant lectins to act as negative controls in our microarrays. Along with the non-binding variants, we developed a lectin displayed on the surface of phage. In the hopes generating more novel lectins, I will describe our current efforts of lectin evolution using phage-displayed GafD. By generating novel tools in lectin microarray technology, we enhance our understanding of the role of carbohydrates on a global scale. / text
8

Hybridization biases of microarray expression data - A model-based analysis of RNA quality and sequence effects

Fasold, Mario 01 July 2013 (has links) (PDF)
Modern high-throughput technologies like DNA microarrays are powerful tools that are widely used in biomedical research. They target a variety of genomics applications ranging from gene expression profiling over DNA genotyping to gene regulation studies. However, the recent discovery of false positives among prominent research findings indicates a lack of awareness or understanding of the non-biological factors negatively affecting the accuracy of data produced using these technologies. The aim of this thesis is to study the origins, effects and potential correction methods for selected methodical biases in microarray data. The two-species Langmuir model serves as the basal physicochemical model of microarray hybridization describing the fluorescence signal response of oligonucleotide probes. The so-called hook method allows to estimate essential model parameters and to compute summary parameters characterizing a particular microarray sample. We show that this method can be applied successfully to various types of microarrays which share the same basic mechanism of multiplexed nucleic acid hybridization. Using appropriate modifications of the model we study RNA quality and sequence effects using publicly available data from Affymetrix GeneChip expression arrays. Varying amounts of hybridized RNA result in systematic changes of raw intensity signals and appropriate indicator variables computed from these. Varying RNA quality strongly affects intensity signals of probes which are located at the 3\' end of transcripts. We develop new methods that help assessing the RNA quality of a particular microarray sample. A new metric for determining RNA quality, the degradation index, is proposed which improves previous RNA quality metrics. Furthermore, we present a method for the correction of the 3\' intensity bias. These functionalities have been implemented in the freely available program package AffyRNADegradation. We show that microarray probe signals are affected by sequence effects which are studied systematically using positional-dependent nearest-neighbor models. Analysis of the resulting sensitivity profiles reveals that specific sequence patterns such as runs of guanines at the solution end of the probes have a strong impact on the probe signals. The sequence effects differ for different chip- and target-types, probe types and hybridization modes. Theoretical and practical solutions for the correction of the introduced sequence bias are provided. Assessment of RNA quality and sequence biases in a representative ensemble of over 8000 available microarray samples reveals that RNA quality issues are prevalent: about 10% of the samples have critically low RNA quality. Sequence effects exhibit considerable variation within the investigated samples but have limited impact on the most common patterns in the expression space. Variations in RNA quality and quantity in contrast have a significant impact on the obtained expression measurements. These hybridization biases should be considered and controlled in every microarray experiment to ensure reliable results. Application of rigorous quality control and signal correction methods is strongly advised to avoid erroneous findings. Also, incremental refinement of physicochemical models is a promising way to improve signal calibration paralleled with the opportunity to better understand the fundamental processes in microarray hybridization.
9

Standardisering av diagnostiska tester genom utveckling av kopplingskemier för sjukdomsspecifika markörer

Gras, Konrad, Giertta Adler, Julia, Jansson, Caroline, Niva, Ted, Nordmark, Adrian January 2016 (has links)
Diagnostiska tester som tillverkas av företaget Thermo Fisher Scientific baseras på microarrayteknologi. Dessa tester används för diagnostik av allergi och autoimmunitet. Genom immobilisering av specifika antigener på en microarrayyta kan man detektera sjukdomsspecifika antikroppar i patientprover. Antikropparna kommer att binda in till de immobiliserade antigenerna och kan sedan detekteras med hjälp av fluorescens. Ett problem som Thermo Fisher Scientific har haft med sina diagnostiska tester är att de ibland visar felaktiga resultat. Detta problem orsakas av ospecifik bindning av antikroppar till antigenerna samt felaktig orientering på antigenerna. Syftet med denna rapport är att presentera förslag på kopplingskemier som kan användas för optimering av diagnostiska tester, med fokus på att eliminera felaktiga resultat. Detta har uppnåtts genom en studie av relevant vetenskaplig litteratur samt kontakt och intervjuer med experter inom olika vetenskapliga områden. För utvärdering av de undersökta kopplingskemierna skapades en lista med kriterier, där kemiernas bindningsspecificitet och standardiserbarhet låg i fokus. Vi identifierade 21 stycken kopplingskemier ifrån litteraturstudier och analyser. Sju av dessa uppfyller alla kriterier som vi och beställaren Thermo Fisher Scientific har satt upp och därför rekommenderas dessa som lämpliga för diagnostiska test. Vi anser att dessa sju är likvärdiga vad gäller nyttan och användbarheten inom diagnostik.
10

Entwicklung von Antikörper-Mikroarray : von Biophysik der Mikrospot-Reaktion bis zur Hochdurchsatzanalyse der Proteine / Development of antibody microarray: from biophysics of microspot reaction to high throughput analysis of proteins

Kusnezow, Wlad January 2006 (has links) (PDF)
Obwohl Protein-Mikroarrays ursprünglich aus dem gut entwickelten und fest etablierten DNA-Pendant entstanden sind, repräsentierte jedoch die Umstellung der Mikroarray-Technik von der DNA- auf die Proteinanalyse aufgrund der enormen physikalisch-chemischen Variabilität der Proteine, deren relativ niedrigen Stabilität und der komplexen Mikrospot-Kinetik eine große technologische Herausforderung. Deshalb setzt das Vorhaben, die Technik der Antikörper–Mikroarrays von ihrem konzeptuellen Zustand ausgehend zu einem robusten, real funktionierenden Werkzeug zu etablieren, nicht nur eine Vielzahl an technologischen Lösungen, sondern auch eine systematische und physikalisch begründete Herangehensweise in dieser technologischen Entwicklung voraus. Das waren im Wesentlichen die zwei wichtigsten, der eigentlichen Entwicklung der Antikörper-Mikroarrays untergeordneten Ziele der Arbeit. Mit dem Ziel, Antikörper-Mikroarrays prinzipiell zu etablieren und eine optimale Immobilisierungschemie für deren Herstellung zu finden, wurden im ersten Teil dieser Arbeit mehrere chemische Beschichtungen von Glasslides optimiert, unterschiedliche Spotting-Bedingungen von Antikörpern für verschiedene Oberflächen getestet und verschiedene Blockierungsverfahren und Strategien zur Aufbewahrung von Slides analysiert. Anschließend wurde eine Reihe von kommerziellen und selbst hergestellten chemisch beschichteten Slides unter den optimierten Bedingungen miteinander verglichen. Als Hauptergebnis dieser Untersuchung wurde die Herstellung der Antikörper-Microarrays etabliert. Unter anderem konnte im Zuge dieser systematischen Analyse gezeigt werden, dass Epoxysilan-modifizierte Oberflächen am besten geeignet sind. Diese Oberfläche ist heutzutage auf dem Gebiet der Protein-Microarrays am weitesten verbreitet und wurde für alle weiteren Studien innerhalb dieser Dissertation verwendet. Die Entwicklung der Antikörper-Mikroarrays in den letzten Jahren demonstrierte erhebliche Schwierigkeiten im Erreichen der nötigen Sensitivität und Reproduzierbarkeit. Um dieser Problematik auf den Grund zu gehen, und die Mikrospot-Kinetik experimentell untersuchen zu können, wurde im Rahmen dieser Arbeit eine modifizierte und für den Fall der Mikrorrays angepasste Variante des Two-Compartment Modells (TCM) entwickelt. TCM ermöglicht auf eine phänomenologische Weise, d.h., dass Diffusionskoeffizienten, Mischintensität oder Dichte der Bindungsstellen nicht bekannt sein müssen, eine quantitative experimentelle Analyse der Mikrospot-Kinetik unter Berücksichtigung von Effekten des Massentransports. Um die phänomenologischen TCM-Werte interpretieren zu können und um den Mechanismus der Mikrospot-Reaktion zu untersuchen, wurden auch andere, für die Mikrospot-Kinetik relevante, klassische Theorien an die Bedingungen der Mikrospot-Reaktion angepasst und mit dem modifizierten TCM mathematisch verbunden. Als das erste in der Mikroarray-Technologie mathematisch-physikalische Werkzeug dieser Art hat die hier entwickelte Theorie ein großes Potential, auch in den anderen verwandten Techniken wie DNA- oder Peptid-Mikroarrays Verwendung zu finden. Außerdem wurde innerhalb dieser Arbeit ein anderes einheitliches theoretisches Modell entwickelt, das eine kinetische Simulation von verschiedenen Reaktionsphasen sowohl für konventionelle als auch für Mikrospot-Immunoassays ermöglicht. Im Rahmen dieser Arbeit konnte für einen typischen Standard-Antikörper-Mikroarray theoretisch und experimentell eine lang andauernde, stark massentransportabhängige Mikrospot-Kinetik beschrieben werden. Es konnte gezeigt werden, dass das Erreichen eines thermodynamischen Gleichgewichts in Mikroarrays wegen eines relativ langsamen Ligandentransports zum Spot eine lange Zeit dauert, je nach Bindungskonstante, Diffusionsgeschwindigkeit und Ligandenkonzentration mehrere Stunden bis hin zu Wochen. In dieser Arbeit wurde ein neues physikalisches Konzept, das dem heutzutage dominierenden Blickwinkel, der sogenannten ambient analyte Theorie, opponierend gegenübersteht, formuliert. Auch konnten viele Konsequenzen fürs Design und die zukünftige Entwicklung dieser relativ neuen Technologie gezogen werden. Als eine logische Folge der massentransportlimitierten Reaktionen ist das Design eines Antikörper-Mikroarray ein kritischer Punkt für die Leistung des Verfahrens. Im Laufe der experimentellen und/oder theoretischen Betrachtungen konnte gezeigt werden, dass eine Reihe allgemeiner Parameter wie Größe eines Spots, Spotting-Muster, Inkubationsgeometrie, Volumen und Konzentration einer Probe, Viskosität des Inkubationspuffers und Mischintensität die Reaktionsraten auf den Spots insgesamt um mehrere Größenordnungen beeinflusst. Ist die maximale Rate des Massentransports in einem Mikroarray-Verfahren gewährleistet, kann dann auch die maximale Bindungsleistung der Spots, die durch die Dichte der Bindungsstellen, Bindungsaffinität, Inkubationszeit und andere relevante Parameter eingestellt wird, erreicht werden. Aber nicht nur in der Inkubationsphase, sondern auch bei den Wasch- und Detektionsschritten sollte die gleiche Liste der Parameter berücksichtigt werden. Durch die Optimierung all dieser Parametern konnte eine deutliche Verbesserung der Sensitivität von Antikörper-Mikroarrays in der Protein-Expressionsanalyse von klinischen Blutproben erzielt werden In einer weiteren Studie zur Analyse von unterschiedlichen Detektionsverfahren konnte die Sensitivität und Reproduzierbarkeit der etablierten Antikörper-Mikroarrays weiter verbessert werden. Eine Reihe unterschiedlicher Markierungssubstanzen mit NHS (N-hydroxysuccinimide) und ULS (universal linkage system) reaktiven Gruppen wurden innerhalb drei Detektionsverfahren untersucht: 1) eine direkte Probenmarkierung mit Fluoreszenzfarbstoffen, 2) Markierung der Probe mit Biotin-Substanzen und nachfolgender Detektion mittels fluoreszenzmarkierten Extravidin und 3) Markierung der Probe mit Fluorescein-Substanzen mit Anti-Fluorescein-Detektion. Aus den Erfahrungen der vorherigen kinetischen Untersuchungen wurde hier vorerst das kinetische Verhalten des Testsystems analysiert und optimale Inkubationsbedingungen festgelegt. Anschließend wurden optimale Konzentrationen all dieser Substanzen für die Markierung von Blutplasma bestimmt. Im Vergleich zur direkten Fluoreszenzmarkierung resultierten sich die indirekten Detektionsverfahren mit Biotin- und Fluorescein-Substanzen in wesentlich besseren Signal-zu-Hintergrund-Verhältnissen. In einer anschließenden Vergleichsanalyse zeigten sich einige Substanzen wie Biotin-ULS oder Fluoresceine-NHS als am besten geeignet für eine Protein-Expressionsanalyse von Blutplasma. Sensitivitäten im femtomolaren Bereich konnten mittels der etablierten Antikörper-Mikroarrays sowohl für eine markierte Antigenmischung als auch für komplexe klinische Proben innerhalb dieser Dissertation erzielt werden. Viele niedrig konzentrierte Proteine wie beispielsweise Zytokine, die normalerweise in einer piko-oder femtomolaren Konzentration im Blut vorliegen, wurden in dieser Arbeit mit sehr hohen Signal-zu-Hintergrund-Verhältnissen detektiert. Das hier beschriebene Verfahren öffnet zusätzliche Möglichkeiten für schnelle, kostengünstige und unbeschränkt erweiterungsfähige Mikrospot-Immunoassays. / Although protein microarrays are superficially similar to DNA microarrays, the conversion of microarray technology to the protein world still represents a big technological challenge because of the enormous physicochemical variability and relatively low stability of proteins as well as complex microspot kinetics. Therefore, the intention of developing the concept of antibody microarray into a really functioning tool requires a multiplicity of technological solutions as well as a systematic and physically justified approach in this technological development. These were essentially the two most important goals while developing antibody microarrays. Aiming to establish antibody microarrays in principle und to find optimal antibody immobilization chemistry, several chemical coatings of glass slides, antibody spotting conditions for different surfaces, different blocking procedures and strategies for slides storage were analysed and optimised in the first part of this dissertation. Subsequently, a set of commercial and home-made chemically coated slides was compared under these optimized conditions. As a main result, manufacturing of antibody microarrays was established. Among other things, the epoxysilan surface was found to be best suitable for fabrication of antibody microarrays in course of this systematic analysis. This kind of chemical coating is nowadays one of the most popular in the protein microarray field and it was also used in all other studies presented here. The development of antibody microarray technology in the last years demonstrated substantial difficulties trying to achieve the required sensitivity and reproducibility. In order to get the bottom of this issue and to be able to examine microspot kinetics experimentally, the so-called two-compartment model (TCM) was modified and adapted for the case of microrrays in this work. TCM enables a quantitative experimental analysis of microspot kinetics with regard to effects of mass transport. It is a phenomenological model, so that one does not need to know density of binding sites or any parameters of mass transport such as diffusion coefficient or mixing intensity. To be able to interpret the phenomenological reaction descriptors of TCM and to investigate reaction mechanism, some relevant theoretical models were also adapted for the case of microspot reaction as well as were mathematically joined with the modified TCM. Our theory is the first mathematical tool of this sort in the microarray technology and it has therefore a lot of potential to be applied also in the other related techniques such as DNA or peptide microarrays. Additionally, another uniform theoretical model was developed in this work. It enables a kinetic simulation of different reaction regimes for the case of conventional as well as microspot immunoassays. Long-lasting and strongly mass transport dependent microspot kinetics was described in this work, both theoretically and experimentally. The attainment of the thermodynamic equilibrium in microarrays may require many hours, days or even weeks due to a relatively slow ligand transport to spots. A new physical concept, which represents the opposite view to the today’s most widespread concept, so called ambient analyte theory, could be formulated in consequence of this investigation. Also, we could draw many consequences for design and future development of this relatively new techique. As a logical consequence of the mass transport limited reactions, proper assay design is cruicial for performance of antibody microarrays. In the course of our experimental and/or theoretical considerations, it was shown that a number of general parameters such as size of a spot, spotting pattern, incubation geometry, volume and concentration of a sample, viscosity of incubation buffer and mixing intensity could altogether affect signal velocity by many orders of magnitude. If the maximum rate of mass transport in a microarray procedure is ensured, then also the maximum binding capacity of spots could be achieved by adjusting such parameters as density of binding sites, binding affinity, incubation time etc. The same factors should be also considered for washing and detection steps in a kinetically relevant design of microarray procedure. Optimizing these parameters, the performance of antibody microarrays as applied for protein profiling of clinical specimens could be strongly improved. A significant improvement of the antibody microarray performance as applied for protein profiling of clinical blood samples could be also achieved in a further study aiming to analyze different detection approaches. A number of labeling substances containing either NHS (N-hydroxysuccinimide) or ULS (universal linkage system) reactive groups was examined within three general detection procedures: 1) direct sample labeling with fluorescence dyes, 2) sample labelling with biotin- containing substances with subsequent detection using fluorescently labeled extravidin and 3) sample labelling with fluorescein-substances with detzection by anti-fluorescein. Based on the experience of the previous kinetic investigations, kinetic behavior of the applied test system was first analyzed in this study to find optimal incubation conditions. Also, optimal blood plasma labelling concentration for every analyzed substance was determined in this study. The indirect detection approaches (biotin/extravidin and fluorescein/anti-fluorescein) resulted in substantially better signal-to-noise ratios as compared to direct fluorescent labeling. Some substances such as biotin-ULS or fluorescein-NHS were found to be best suitable for microarray-based protein profiling of blood plasma. Using the established antibody microarrays, sensitivities in the fM range could be attained in this dissertation both for a labeled mix of antigens as well as for complex clinical samples. Many low abundant proteins as for example cytokines, which are normally present in a pM-fM concentration in the blood, was detected in this work with very high signal-to-noise ratios. The approach described here opens additional possibilities for fast, economical and unrestricted multiplexing microspot immunoassays.

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