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Poly-and oligothiophenes: Optical probes for multimodal fluorescent assessment of biological processes
Linköpings universitet, Institutionen för fysik, kemi och biologi, Kemi. Linköpings universitet, Tekniska fakulteten.
2015 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

One interesting class of molecules in the research field of imaging biological processes is luminescent conjugated polythiophenes, LCPs. These fluorescent probes have a flexible backbone consisting of repetitive thiophene units. Due to this backbone, the probes possess unique abilities to give rise to different spectral signatures depending on their target and environment. LCPs are a polydispersed material meaning there is an uneven distribution of lengths of the probe. Recently, monodispersed chemically well-defined material denoted luminescent conjugated oligothiophenes, LCOs, with an exact number of repetitive units and distinct sidechain functionalities along the backbone has been developed. LCOs have the advantages of being smaller which leads to higher ability to cross the blood brain barrier. The synthesis of minor chemical alterations is also more simplified due to the well-defined materials.

During my doctoral studies I have used both LCPs and LCOs to study biological processes such as conformational variation of protein aggregates in prion diseases and cellular uptake in normal cells and cancer cells. The research has generally been based on the probes capability to emit light upon irradiation and the interaction with their targets has mainly been assessed through variations in fluorescence intensity, emission-and excitation profiles and fluorescence lifetime decay. These studies verified the utility of LCPs and LCOs for staining and discrimination of both prion strains and cell phenotypes. The results also demonstrated the pronounced influence minor chemical modifications have on the LCO´s staining capacity.

Ort, förlag, år, upplaga, sidor
Linköping: Linköping University Electronic Press, 2015. , s. 54
Serie
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1693
Nationell ämneskategori
Kemi Cell- och molekylärbiologi
Identifikatorer
URN: urn:nbn:se:liu:diva-121815DOI: 10.3384/diss.diva-121815ISBN: 978-91-7685-986-5 (tryckt)OAI: oai:DiVA.org:liu-121815DiVA, id: diva2:859490
Disputation
2015-11-06, Planck, Fysikhuset, Campus Valla, Linköping, 10:15 (Svenska)
Opponent
Handledare
Tillgänglig från: 2015-10-07 Skapad: 2015-10-07 Senast uppdaterad: 2018-04-25Bibliografiskt granskad
Delarbeten
1. Multimodal fluorescene microscopy of prion strain specific PrP deposits stained by thiophene-bassed amyloid ligands
Öppna denna publikation i ny flik eller fönster >>Multimodal fluorescene microscopy of prion strain specific PrP deposits stained by thiophene-bassed amyloid ligands
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2014 (Engelska)Ingår i: Prion, ISSN 1933-6896, E-ISSN 1933-690X, Vol. 8, nr 4, s. 319-329Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies.

Ort, förlag, år, upplaga, sidor
Taylor & Francis, 2014
Nationell ämneskategori
Kemi Naturvetenskap
Identifikatorer
urn:nbn:se:liu:diva-106792 (URN)10.4161/pri.29239 (DOI)000348376000006 ()
Tillgänglig från: 2014-05-23 Skapad: 2014-05-23 Senast uppdaterad: 2018-04-25Bibliografiskt granskad
2. Differential vital staining of normal fibroblasts and melanoma cells by an anionic conjugated polyelectrolyte
Öppna denna publikation i ny flik eller fönster >>Differential vital staining of normal fibroblasts and melanoma cells by an anionic conjugated polyelectrolyte
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2015 (Engelska)Ingår i: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 87, nr 3, s. 262-272Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes.

Ort, förlag, år, upplaga, sidor
Wiley: 12 months, 2015
Nyckelord
Conjugated polyelectrolyte; Fibroblast; Fluorescence; Luminescent conjugated polythiophene; Melanoma; Photoinduced toxicity
Nationell ämneskategori
Strukturbiologi Cell- och molekylärbiologi
Identifikatorer
urn:nbn:se:liu:diva-115887 (URN)10.1002/cyto.a.22627 (DOI)000349984200009 ()25605326 (PubMedID)2-s2.0-84923259526 (Scopus ID)
Tillgänglig från: 2015-03-23 Skapad: 2015-03-23 Senast uppdaterad: 2018-09-14
3. Cell Type Related Differences in Staining with Pentameric Thiophene Derivatives
Öppna denna publikation i ny flik eller fönster >>Cell Type Related Differences in Staining with Pentameric Thiophene Derivatives
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2014 (Engelska)Ingår i: Cytometry Part A, ISSN 1552-4922, E-ISSN 1552-4930, Vol. 85A, nr 7, s. 628-635Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Fluorescent compounds capable of staining cells selectively without affecting their viability are gaining importance in biology and medicine. Recently, a new family of optical dyes, denoted luminescent conjugated oligothiophenes (LCOs), has emerged as an interesting class of highly emissive molecules for studying various biological phenomena. Properly functionalized LCOs have been utilized for selective identification of disease-associated protein aggregates and for selective detection of distinct cells. Herein, we present data on differential staining of various cell types, including cancer cells. The differential staining observed with newly developed pentameric LCOs is attributed to distinct side chain functionalities along the thiophene backbone. Employing flow cytometry and fluorescence microscopy we examined a library of LCOs for stainability of a variety of cell lines. Among tested dyes we found promising candidates that showed strong or moderate capability to stain cells to different extent, depending on target cells. Hence, LCOs with diverse imidazole motifs along the thiophene backbone were identified as an interesting class of agents for staining of cancer cells, whereas LCOs with other amino acid side chains along the backbone showed a complete lack of staining for the cells included in the study. Furthermore, for p-HTMI,a LCO functionalized with methylated imidazole moieties, the staining was dependent on the p53 status of the cells, indicating that the molecular target for the dye is a cellular component regulated by p53. We foresee that functionalized LCOs will serve as a new class of optical ligands for fluorescent classification of cells and expand the toolbox of reagents for fluorescent live imaging of different cells.

Ort, förlag, år, upplaga, sidor
John Wiley & Sons, 2014
Nyckelord
cancer stem cells; luminescent conjugated oligothiophenes; fluorescent probes
Nationell ämneskategori
Klinisk medicin Kemi Medicinsk bioteknologi Data- och informationsvetenskap
Identifikatorer
urn:nbn:se:liu:diva-109171 (URN)10.1002/cyto.a.22437 (DOI)000338007700010 ()24500794 (PubMedID)
Tillgänglig från: 2014-08-12 Skapad: 2014-08-11 Senast uppdaterad: 2018-01-11Bibliografiskt granskad
4. An imidazole functionalized pentameric thiophene displays different staining patterns in normal and malignant cells
Öppna denna publikation i ny flik eller fönster >>An imidazole functionalized pentameric thiophene displays different staining patterns in normal and malignant cells
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2015 (Engelska)Ingår i: Frontiers in Chemistry, E-ISSN 2296-2646, Vol. 3, artikel-id 58Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Molecular tools for fluorescent imaging of cells and their components are vital for understanding the function and activity of cells. Here, we report an imidazole functionalized pentameric oligothiophene, p-HTIm, that can be utilized for fluorescent imaging of cells. p-HTIm fluorescence in normal cells appeared in a peripheral punctate pattern partially co-localized with lysosomes, whereas a one-sided perinuclear Golgi associated localization of the dye was observed in malignant cells. The uptake of p-HTIm was temperature dependent and the intracellular target was reached within 1 h after staining. The ability of p-HTIm to stain cells was reduced when the imidazole side chain was chemically altered, verifying that specific imidazole side-chain functionalities are necessary for achieving the observed cellular staining. Our findings confirm that properly functionalized oligothiophenes can be utilized as fluorescent tools for vital staining of cells and that the selectivity towards distinct intracellular targets are highly dependent on the side-chain functionalities along the conjugated thiophene backbone.

Ort, förlag, år, upplaga, sidor
Frontiers Media S.A., 2015
Nyckelord
Oligothiophenes, fluorescence, cells, imaging, imidazole
Nationell ämneskategori
Klinisk medicin Kemi Medicinsk bioteknologi
Identifikatorer
urn:nbn:se:liu:diva-121813 (URN)10.3389/fchem.2015.00058 (DOI)000373364600001 ()
Anmärkning

Vid tiden för disputation förelåg publikationen som manuskript

Funding agencies:  Swedish Foundation for Strategic Research; GeCONil [POIG.02.03.01-24-099/13]; ERC from the European Research Council

Tillgänglig från: 2015-10-07 Skapad: 2015-10-07 Senast uppdaterad: 2017-12-01Bibliografiskt granskad

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