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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.
11

In situ molecular profilling of the microenvironment of breast carcinoma

Kaira, Mustapha January 2015 (has links)
High stromal PDGF receptor B expression was shown to have strong prognostic value in a studyinvolving over 600 breast cancer patients however, the molecular role of the receptor in tumordevelopment remains unclear. In this project we studied the spatial distribution and expressionlevels of a panel genes and markers associated with PDGF signaling, in breast cancer tumormicroenvironment (TME) using a newly developed technique -in situ sequencing. The techniquerelies on padlock probes which we validated with corresponding RNA sequencing, microarray,and immunohistochemistry data. Our results showed that high PDGF receptor B mRNA colocalizedwith markers of two pathways, TGFβ and Hedgehog signaling; this suggests that theymight contribute to the PDGF-receptor B-driven tumor growth. We also showed that stromalPDGF signaling is stimulated predominantly by tumor cells. Finally, further expression profilingof each individual gene revealed that CXCL14 was mainly expressed in the stroma, ACTA2expression was enriched in the tumor/stroma boundary while the stem-cell marker, OCT3, wasexpressed in the interior of the tumor cells.
12

Nyckelfri V&M : Ett elektroniskt hänglås med Bluetooth / Keyless V&M : An electronic padlock with Bluetooth

Valeur, Allan, Mood, Lars January 2023 (has links)
Detta är ett kandidatexamensarbete i mekatronik där ett elektroniskt hänglås med Bluetooth designas, konstrueras och utvärderas. Forskningsfrågorna som besvaras lyder enligt följande: Hur kan de mekaniska delarna utformas och integreras med elektroniken för en enkel och pålitlig konstruktion av ett elektroniskt hänglås? Vilka användningsområden är fördelaktiga för ett elektroniskt hänglås med Bluetooth? En prototyp av hänglåset designas i CAD och produceras sedan med hjälp av 3D-printing. Den elektroniska kretsen styrs av en Arduino Nano, som kontrollerar en servomotor, vilken i sin tur vrider låsmekanismen. Tester utförs på prototypens batteritid, Bluetooth-räckvidd och mekanism. Testerna visar att prototypen har en batteritid på 27 timmar, en- Bluetooth-räckvidd på 8 meter och en mekanism som fungerar felfritt. Utöver detta identifieras en rad brister angående låsets säkerhet och funktionalitet. Således visar undersökningen att ett elektroniskt hänglås med Bluetooth-funktion kan konstrueras på ett enkelt och pålitligt sätt, men slutsatsen dras att dess användnings områden begränsas tilllåsning under korta perioder där bekvämlighet är högre prioriterat än säkerhet. / This is a bachelor’s thesis in mechatronics where an electronic padlock is designed, constructed and evaluated. The research questions for the thesis are: How can the mechanical parts be designed and integrated with the electronics to create a simple and reliable construction of an electronic padlock? What use cases are advantageous for an electronic padlock using Bluetooth? A prototype of the padlock is designed in CAD and produced using 3D-printing. The electronic circuit is controlled by an Arduino Nano, which controls a servo motor, which in turn operates the locking mechanism. Tests are conducted on the prototype's battery, Bluetooth-range and mechanism. The tests show that the prototype has a battery-life of 27 hours, a Bluetooth range of 8 meters and a mechanism that works without fail. Additionally a number of fl aws are identified with the prototype, both regarding its security and functionality. The research thus shows that an electronic padlock using Bluetooth is possible to create in a simple and reliable way, but the conclusion is drawn that its use cases are limited to locking things during short periods, where user comfort is prioritized above security.
13

Genotyping RNA and DNA using padlock probes

Antson, Dan-Oscar January 2001 (has links)
Novel techniques are needed to investigate the genetic variation revealed in the first draft of the human genome sequence. Padlock probes are recently developed reagents, suitable for detecting single-nucleotide variations of DNA and RNA in situ or in solution. The probes are oligonucleotides of about 70-140 nucleotides that can be circularized by ligation in the presence of a correct target sequence. Standard chemical synthesis of padlock probes is difficult due to the requirement for intact 5' and 3' ends of these long oligonucleotides. A novel PCR-based method is presented in this thesis, whereby longer, densely labeled padlock probes can be made as compared to conventional chemical synthesis. PCR-generated padlock probes produced a stronger signal and a more resolved staining pattern, compared to chemically synthesized probes in fluorescence in situ analysis of an alpha-satellite sequence variant present in human chromosomes 13 and 21. Padlock probes used for in situ analysis of metaphase chromosomes had an optimal length of 140 nucleotides. They were used to identify individual chromosomes 7 and 15, and to follow the transmission of chromosome homologues for two consecutive generations. The specificity of the padlock probes to detect single copy genes in genomic DNA samples was demonstrated by detecting a single-nucleotide mutation in the ATP7B gene. It has not previously been known if T4 DNA ligase can be used for RNA sequence analysis. In this thesis, it is demonstrated that T4 DNA ligase can be used for distinguishing single-nucleotide RNA sequence variants. Reaction conditions were defined where most mismatches could be discriminated by a factor of 80 and all mismatches by a factor of at least 20. Under these conditions padlock probes could detect and distinguish RNA sequence variants with ligation efficiency almost as high as on the corresponding DNA sequence. A detailed study of the parameters influencing RNA-templated DNA ligation revealed that DNA ligation on RNA templates proceeds at a much slower rate compared to the same reaction on DNA, and that a molar excess of enzyme is required. Furthermore, the ligation reaction is inhibited by high concentrations of the cofactor ATP and NaCl. The work presented in this thesis demonstrates that PCR-generated padlock probes can detect and distinguish single-nucleotide variation in both RNA and DNA.
14

Ligation-mediated Molecular Analysis of Influenza Subtypes, Splicing and Protein Glycosylation

Conze, Tim January 2010 (has links)
Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples. We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I). Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II). Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III). This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.
15

Validation and optimization of multiplexInSitu PLA for signalling pathway analysis

Sinha, Tanay Kumar January 2021 (has links)
With the advent of Tyrosine kinase inhibitors (TKI) as a therapy for Chronic myeloid Leukemia (CML), the patients now enjoy a life expectancy close to that of the general population. But some patients do get unresponsive to the TKI treatment over time due to several mutations in the kinase domain of the BCR-ABL fusion protein, which further leads to activation of multiple signaling cascades within the leukemic cell, helping it survive and proliferate. This project validates and optimizes a new method of In situ PLA that incorporates the usage of different padlocks and template oligos. Multiple cross-reactivity tests and interaction assays in multiple cancer cell lines will further optimize this system as a robust multiplex protein-protein interaction detection tool. Proteins associated with the MAP-K, PI3-K, and Jak-STAT signaling pathways were the main detection targets.
16

Single Molecule Detection : Microfluidic Automation and Digital Quantification

Kühnemund, Malte January 2016 (has links)
Much of recent progress in medical research and diagnostics has been enabled through the advances in molecular analysis technologies, which now permit the detection and analysis of single molecules with high sensitivity and specificity. Assay sensitivity is fundamentally limited by the efficiency of the detection method used for read-out. Inefficient detection systems are usually compensated for by molecular amplification at the cost of elevated assay complexity. This thesis presents microfluidic automation and digital quantification of targeted nucleic acid detection methods based on padlock and selector probes and rolling circle amplification (RCA). In paper I, the highly sensitive, yet complex circle-to-circle amplification assay was automated on a digital microfluidic chip. In paper II, a new RCA product (RCP) sensing principle was developed based on resistive pulse sensing that allows label free digital RCP quantification. In paper III, a microfluidic chip for spatial RCP enrichment was developed, which enables the detection of RCPs with an unprecedented efficiency and allows for deeper analysis of enriched RCPs through next generation sequencing chemistry. In paper IV, a smart phone was converted into a multiplex fluorescent imaging device that enables imaging and quantification of RCPs on slides as well as within cells and tissues. KRAS point mutations were detected (i) in situ, directly in tumor tissue, and (ii) by targeted sequencing of extracted tumor DNA, imaged with the smart phone RCP imager. This thesis describes the building blocks required for the development of highly sensitive low-cost RCA-based nucleic acid analysis devices for utilization in research and diagnostics.
17

Genetic Analyses using Rolling Circle or PCR Amplified Padlock Probes

Banér, Johan January 2003 (has links)
<p>Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis.</p><p>The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization.</p><p>Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.</p>
18

Genetic Analyses using Rolling Circle or PCR Amplified Padlock Probes

Banér, Johan January 2003 (has links)
Padlock probes are useful in a variety of genetic applications, some of which require that the probes are amplified in order to generate detectable signals. Two general padlock amplification methods, RCA and PCR, are discussed in this thesis. The isothermal rolling circle amplification (RCA) mechanism is described in detail as well as how a target strand affects primer extension. A mechanism to resolve the topological constraint imposed by the target strand, to which a padlock probe has been linked, is also discussed. We also present a more powerful amplification technique, termed serial circle amplification, which provides a highly precise tool for nucleic acid studies. Rolling circle products are digested to unit lengths, and each monomer converted to new circular oligonucleotides that can serve as templates in consecutive rounds of RCA. The final products are single-stranded DNA molecules, readily available for hybridization-based detection, for instance using molecular beacons or array hybridization. Padlock probes have the potential to be combined in large numbers for parallel gene analysis. A significant improvement of the level of multiplexed genotyping is presented using padlock probes and a molecular inversion strategy. Padlock probes containing common primer sequences along with locus-specific tag sequences were combined in multiplexed ligation reactions. After exonucleolytic selection for circular molecules, the probes were cleaved at uracil residues situated between the primer sequences, which facilitated release from the genomic DNA. A single PCR primer pair amplified all molecularly inverted probes, and the products were finally sorted on microarrays for simultaneous readout. Up to 1,500 genotypes could be detected in parallel, with sufficient signal strength for further scale-up. Finally, an application of the same parallel genotyping strategy is described where a set of padlock probes was used to study tumor induced immune responses. The distribution of TCR Vβ transcripts in tumor infiltrating T-cells and in normal control tissues were investigated in a microarray format.
19

Molecular Diagnostics Using Volume-Amplified Magnetic Nanobeads : Towards the Development of a Novel Biosensor System

Strömberg, Mattias January 2009 (has links)
Micro- or nanometer sized magnetic particles (beads) currently have a vast range of life science applications in, for example, bioseparation techniques, cancer therapy, development of contrast agents and biosensing techniques. In the latter field, magnetic beads offer several unique advantages, including minimal background signals, physical and chemical stability and low manufacturing costs. Because of these properties, magnetic biosensing techniques are potential candidates for low-cost, easy-to-use molecular diagnostic devices. This doctoral thesis focuses mainly on the proof of principle and further development of a new magnetic biosensor platform for detection of DNA targets, a potential candidate for a new generation of low-cost, easy-to-use diagnostic devices: the Volume-Amplified Magnetic Nanobead Detection Assay (VAM-NDA). The VAM-NDA principle combines target recognition by padlock probe ligation followed by rolling circle amplification (RCA) of the reacted probes with changes in Brownian relaxation behaviour of magnetic nanobeads (typically ~100 nm in diameter) induced by a change in hydrodynamic bead volume. More specifically, the RCA products (coils, typically ~1 μm in diameter) are detected magnetically by adding magnetic beads tagged with detection probes complementary to part of the repeating RCA-coil sequence. Thus, depending on the target concentration, a certain quantity of beads binds to the coils by base-pair hybridisation (bead immobilisation), resulting in a dramatic bead volume increase, which is then detected by measuring the complex magnetisation spectrum. Use of a commercial SQUID magnetometer for measuring complex magnetisation resulted in a detection limit in the low pM range for DNA targets with excellent quantification accuracy. Simultaneous multiplexing was also evaluated. The stability and aging of typical commercial ferrofluids (suspensions of magnetic beads) were investigated by measuring the complex magnetisation of and interbead interactions in oligonucleotide-functionalised ferrofluids. In summary, the bead surface characteristics were found to have a strong impact on the measured dynamic magnetic properties.
20

Readout Strategies for Biomolecular Analyses

Göransson, Jenny January 2008 (has links)
This thesis describes three readout formats for molecular analyses. A common feature in all works is probing techniques that upon specific target recognition ideally results in equimolar amounts of DNA circles. These are then specifically amplified and detected using any of the techniques presented herein. The first paper presents a method that enables homogeneous digital detection and enumeration of biomolecules, represented as fluorescence-labelled DNA macromolecules. This method offers precise measurements to be performed with a wide linear dynamic range. As an application, two closely related bacterial species were selectively detected. The second paper further investigates and optimizes the properties of the technique presented in paper one. The third paper demonstrates a platform that enables simultaneous quantitative analysis of large numbers of biomolecules. The array format and decoding scheme together propose a digital strategy for decoding of biomolecules. The array and the decoding procedure were characterized and evaluated for gene copy-number measurements. The fourth paper examines a new strategy for non-optical measurements of biomolecules. Characteristics of this technique are investigated, and compared to its optical equivalent, fluorescence polarization.

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