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Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Matematisk-datavetenskapliga sektionen, Institutionen för informationsteknologi, Avdelningen för visuell information och interaktion. Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Matematisk-datavetenskapliga sektionen, Institutionen för informationsteknologi, Bildanalys och människa-datorinteraktion.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för immunologi, genetik och patologi, Molekylära verktyg.
Vise andre og tillknytning
2015 (engelsk)Inngår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, s. 12317:1-10, artikkel-id 12317Artikkel i tidsskrift (Fagfellevurdert) Published
sted, utgiver, år, opplag, sider
2015. Vol. 5, s. 12317:1-10, artikkel-id 12317
HSV kategori
Forskningsprogram
Datoriserad bildbehandling
Identifikatorer
URN: urn:nbn:se:uu:diva-260286DOI: 10.1038/srep12317ISI: 000358358900001PubMedID: 26202090OAI: oai:DiVA.org:uu-260286DiVA, id: diva2:847762
Forskningsfinansiär
EU, FP7, Seventh Framework Programme, 278568EU, FP7, Seventh Framework Programme, 259796Swedish Research CouncilTilgjengelig fra: 2015-07-23 Laget: 2015-08-18 Sist oppdatert: 2018-02-27bibliografisk kontrollert
Inngår i avhandling
1. Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay
Åpne denne publikasjonen i ny fane eller vindu >>Analysis of signaling pathway activity in single cells using the in situ Proximity Ligation Assay
2016 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

A cell that senses signals from its environment uses proteins for signal transduction via post translational modifications (PTMs) and protein- protein interactions (PPIs) from cell membrane into the nucleus where genes controlling cell proliferation, differentiation and apoptosis can be turned on or off, i.e. changing the phenotype or fate of the cell. Aberrations within such proteins are prone to cause diseases, such as cancer. Therefore, it is important so study aberrant signaling to be able to understand and treat diseases.

In this thesis, signaling aberrations of PTMs and PPIs were analyzed with the use of the in situ proximity ligation assay (in situ PLA), and the thesis also contain method development of rolling circle amplification (RCA), which is the method used for signal amplification of in situ PLA reaction products.

Paper I considers the integrity of RCA products. Here, the aim was to generate a smaller and more compact RCA product, for more accurate either visual or automated analysis. This was achieved with the use of an additional so called compaction oligonucleotide that during RCA was able to bind and pull segments of RCA products closer together. The compaction oligonucleotide served to increase the signal to noise ratio and decrease the number of false positive signals.

The crosstalk between the Hippo and TGFβ signaling pathways were studied in paper II. Activity of the Hippo signaling pathway is regulated by cell density sensing and tissue control. We found differences in amounts and localization of interactions between the effector proteins of the two pathways depending on cell density and TGFβ stimulation.

In paper III the NF-кB signaling pathway constitutively activated in chronic lymphocytic leukemia (CLL) was studied. A 4 base-pair frameshift deletion within the NFKBIE gene, which encodes the negative regulator IкBε, was found among 13 of a total 315 cases by the use of targeted deep sequencing. We found reduced levels of IкBε protein, decreased p65 inhibition, and increased phosphorylation, along with increased nuclear localization of p65 in NFKBIE deleted cases compared to healthy cases.

Crosstalk between the Hippo and Wnt signaling pathway are studied within paper IV. Here, we found differences in cellular localization of TAZ/β-catenin interactions depending on colon cancer tumor stage and by further investigate Hippo/WNT crosstalk in cell line model systems we found an increase of complex formations involved in the crosstalk in sparse growing HEK293 cells compared to dense growing cells. Also, active WNT3a signaling was affected by cell density. Since cell density showed to have a big effect on Hippo/WNT crosstalk we continued to investigated the effect of E-cadherin, which has a function in cell junctions and maintenance of epithelial integrity on Hippo/WNT crosstalk. Interestingly, we found that E-cadherin is likely to regulate Hippo/WNT crosstalk.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2016. s. 45
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1202
Emneord
cell signaling, Wnt, Hippo, TGFB
HSV kategori
Forskningsprogram
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-281716 (URN)978-91-554-9529-9 (ISBN)
Disputas
2016-05-20, BMC, B41, Husargatan 3, Uppsala, 13:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2016-04-28 Laget: 2016-03-29 Sist oppdatert: 2016-05-12
2. Image Analysis and Deep Learning for Applications in Microscopy
Åpne denne publikasjonen i ny fane eller vindu >>Image Analysis and Deep Learning for Applications in Microscopy
2016 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Quantitative microscopy deals with the extraction of quantitative measurements from samples observed under a microscope. Recent developments in microscopy systems, sample preparation and handling techniques have enabled high throughput biological experiments resulting in large amounts of image data, at biological scales ranging from subcellular structures such as fluorescently tagged nucleic acid sequences to whole organisms such as zebrafish embryos. Consequently, methods and algorithms for automated quantitative analysis of these images have become increasingly important. These methods range from traditional image analysis techniques to use of deep learning architectures.

Many biomedical microscopy assays result in fluorescent spots. Robust detection and precise localization of these spots are two important, albeit sometimes overlapping, areas for application of quantitative image analysis. We demonstrate the use of popular deep learning architectures for spot detection and compare them against more traditional parametric model-based approaches. Moreover, we quantify the effect of pre-training and change in the size of training sets on detection performance. Thereafter, we determine the potential of training deep networks on synthetic and semi-synthetic datasets and their comparison with networks trained on manually annotated real data. In addition, we present a two-alternative forced-choice based tool for assisting in manual annotation of real image data. On a spot localization track, we parallelize a popular compressed sensing based localization method and evaluate its performance in conjunction with different optimizers, noise conditions and spot densities. We investigate its sensitivity to different point spread function estimates.

Zebrafish is an important model organism, attractive for whole-organism image-based assays for drug discovery campaigns. The effect of drug-induced neuronal damage may be expressed in the form of zebrafish shape deformation. First, we present an automated method for accurate quantification of tail deformations in multi-fish micro-plate wells using image analysis techniques such as illumination correction, segmentation, generation of branch-free skeletons of partial tail-segments and their fusion to generate complete tails. Later, we demonstrate the use of a deep learning-based pipeline for classifying micro-plate wells as either drug-affected or negative controls, resulting in competitive performance, and compare the performance from deep learning against that from traditional image analysis approaches. 

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2016. s. 76
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1371
Emneord
Machine learning, Deep learning, Image analysis, Quantitative microscopy, Bioimaging
HSV kategori
Forskningsprogram
Datoriserad bildbehandling
Identifikatorer
urn:nbn:se:uu:diva-283846 (URN)978-91-554-9567-1 (ISBN)
Disputas
2016-06-09, 2446, ITC, Lägerhyddsvägen 2, Hus 2, Uppsala, 10:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2016-05-18 Laget: 2016-04-14 Sist oppdatert: 2016-06-01bibliografisk kontrollert
3. Development of DNA-based methods for analysis of protein interactions
Åpne denne publikasjonen i ny fane eller vindu >>Development of DNA-based methods for analysis of protein interactions
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

In situ proximity ligation assay (PLA) is a method for detection of protein interactions, post-translational modifications (PTMs) and individual proteins that allows information about their localization in a cell or tissue to be extracted. The method is based on oligonucleotide-conjugated antibodies (proximity probes) that upon binding of two epitopes in close proximity give rise to an amplifiable DNA circle. Rolling circle amplification (RCA) is used to create a DNA bundle of over a thousand repeats to which fluorescently labeled detection oligonucleotides are hybridized. This thesis is focused on improving the existing in situ PLA method and on developing new approaches for detection of proteins, protein-protein interactions and PTMs in situ in cells and tissues.

In paper I, a new enzyme-independent method capable of in situ detection of protein-protein interactions was developed. The method combined the proximity requirement of in situ PLA and the amplification of hybridization chain reaction (HCR) creating a proximity-dependent initiation of hybridization chain reaction (proxHCR). Circumventing the need for enzymes resulted in a cost-efficient method that is less sensitive to storing conditions.

Paper II addresses the problem of irregularly formed RCA products that can appear to be split into several fluorescent objects. A compaction oligonucleotide system was designed to crosslink the DNA bundle with itself and thereby reduce the size and increase the brightness of each individual RCA product.

In paper III, the conventional in situ PLA was redesigned to increase the detection efficiency of protein interactions and PTMs in situ. The new set of proximity probes was designed to have circularization oligonucleotides incorporated that were unfolded through enzymatic digestion. The UnFold in situ PLA was able to generate more signals and had a higher sensitivity than the conventional in situ PLA.

In paper IV, an oligonucleotide system able to generate signals for individual proteins (A or B) and their interaction (A and B) in a molecular Boolean (MolBoolean) protein analysis was designed. The MolBoolean design was able to generate signals detecting both individual proteins and their interaction in situ.  

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2018. s. 47
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 245
Emneord
In situ proximity ligation assay (PLA), rolling circle amplification (RCA), Hybridization chain reaction (HCR), proxHCR, Oligonucleotide design, Protein-protein interactions, Post-translational modifications.
HSV kategori
Forskningsprogram
Farmaceutisk vetenskap; Molekylär cellbiologi
Identifikatorer
urn:nbn:se:uu:diva-340078 (URN)978-91-513-0222-5 (ISBN)
Disputas
2018-03-15, A1:107a, BMC, Husargatan 3, Uppsala, 13:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2018-02-21 Laget: 2018-01-25 Sist oppdatert: 2018-03-07

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