| 1. |
- Fu, Ying, 1964-, et al.
(författare)
-
Endocytic pathway of vascular cell adhesion molecule 1 in human umbilical vein endothelial cell identified in vitro by using functionalized nontoxic fluorescent quantum dots
- 2019
-
Ingår i: Sensors and actuators. B, Chemical. - : Elsevier B.V.. - 0925-4005 .- 1873-3077. ; 297
-
Tidskriftsartikel (refereegranskat)abstract
- Studies about vascular cell adhesion molecule 1 (VCAM1) in tumor growth, metastasis, and angiogenesis suggest that targeting VCAM1 expression is an attractive strategy for diagnosis and anti-tumor therapy. However, the endocytic pathway of VCAM1 in vascular cells has not been well characterized. In this study we visualize the endocytic pathway of tumor necrosis factor α (TNFα) induced VCAM1 in human umbilical vein endothelial cell (HUVEC) in vitro using 5-carboxyfluorescein labeled VCAM1 binding peptides and fluorescent water-dispersible 3-mercaptopropionic acid (3MPA)-coated CdSe-CdS/Cd0.5Zn0.5S/ZnS core–multishell nontoxic quantum dots (3MPA-QDs) functionalized with VCAM1 binding peptides. Clear key in vitro observations are as follows: (a) 3MPA-QDs functionalized with VCAM1 binding peptides, denoted as VQDs, adhered and aggregated cumulatively to cell membrane around 2 h after VQD deposition to cell culture medium and were found in lysosomes in TNFα-treated HUVECs approximately 24 h after VQD deposition; (b) VQDs remained in TNFα-treated HUVECs for the whole 16 days of the experimental observation period; (c) quite differently, 3MPA-QDs were endocytosed then exocytosed by HUVECs via endosomes in about 24–48 h after 3MPA-QD deposition. Our study suggests that VCAM1 molecules, initially expressed on cell membrane induced by TNFα treatment, are internalized into lysosomes. This provides a novel means to deliver materials to lysosomes such as enzyme replacement therapy. Moreover, our meticulous sensing methodology of devising fluorescent nontoxic QDs advances biosensing technique for studying cellular activities in vitro and in vivo. © 2019 The Authors
|
|
| 2. |
- Jess, David Unnersjö
(författare)
-
High-resolution Imaging of Cleared and Expanded Kidney Tissue Samples
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The kidney is one of the most important and complex organs in the humanbody with the task of filtering hundreds of litres of blood daily. It is responsiblefor the salt and acid/base balance in the body, as well as secretinghormones important for red blood cell production and blood pressure regulation. Kidney disease is one of the fastest growing causes of death in the modern world, and this motivates extensive research for better understandingthe function of the kidney in both health and disease. Kidney failure or end stage renal disease (ESRD) is irreversible and requires treatment with dialysisor transplantation. Some of the most important cellular structures for blood filtration in the kidney are of very small dimensions (below 200 nanometers), and thus electron microscopy has previously been the only method with high enough resolution to study the morphology and topology of these minute structures. In three studies included in this thesis, we show that the finest elements of the kidney can now be resolved using different light microscopy techniques. In study 1, we show that by combining optical clearing with STED microscopy, protein localizations in the slit diaphragm of the kidney can be resolved, with widths around 75 nanometers. In study 3, a novel sample preparation method, expansion microscopy, is utilized to isotropically expand kidney tissue samples in space. Expansion improves the effective resolution by a factor of 5, making it possible to resolve podocyte foot processes and the slit diaphragmusing diffraction-limited confocal microscopy. We also show that by combining expansion microscopy and STED microscopy, the effective resolution can be improved even further (<20 nm). In our most recent work, study 5, we apply a simplified, moderate tissue swelling protocol which together with optimization of the confocal imaging provides sufficient resolution to resolve foot processes and parts of the filtration barrier. This new protocol is fast and technically simple, making it ideal for routine use, such as for future clinical pathology. In collaboration with kidney researchers, we have applied both STED microscopy and expansion microscopy to various disease models, showing that these tools can be used to both visualize and quantify pathologies occurring in different parts of the glomerular filtration barrier (GFB). In study 2, STED microscopy in combination with optical clearing is used to study the localization of Coro2b in secondary foot processes in both mouse and human tissue. In two ongoing studies with preliminary results presented in the thesis, we use STED microscopy and optical clearing to study the pathogenesis of focal segmental glomerulosclerosis (FSGS) by the use of genetic mouse models. Based on STED images, we extract different morphological parameters from foot processes and the glomerular filtration barrier (GFB) at different stages of the disease. In study 4, we apply a tissue expansion protocol to answer questions about the phenotype seen in podocytes where the mediator complex subunit 22 (Med22) is inactivated. By inactivating Med22 in a transgenic mouse line with cytosolic expression of tdTomato in podocytes, we saw strong indications that the vesicle-like structures seen in EM micrographs were indeed intracellular vesicles and not dilated sub-podocyte space. In summary, the work presented in this thesis has contributed to the development of a new toolbox for imaging renal ultra-structure using light microscopy, a field previously reserved for electron microscopy.
|
|
