Since the publication of the complete sequence of the human genome in 2003 there has been great interest in exploring the functions of the proteins encoded by the genes. To reveal the function of each and every protein, investigation of protein localization at the subcellular level has become a central focus in this research area, since the localization and function of a protein is closely related. The objective of the studies presented in this doctoral thesis was to systematically explore the human proteome at the subcellular level using bioimaging and to develop techniques for validation of the results obtained. A common imaging technique for protein detection is immunofluorescence (IF), where antibodies are used to target proteins in fixated cells. A fixation protocol suitable for large-scale IF studies was developed and optimized to work for a broad set of proteins. As the technique relies on antibodies, validation of their specificity to the target protein is crucial. A platform based on siRNA gene silencing in combination with IF was set-up to evaluate antibody specificity by quantitative image analysis before and after suppression of its target protein. As a proof of concept, the platform was then used for validation of 75 antibodies, proving it to be applicable for validation of antibodies in a systematic manner. Because of the fixation, there is a common concern about how well IF data reflects the in vivo subcellular distribution of proteins. To address this, 500 proteins were tagged with green fluorescent protein (GFP) and used to compare protein localization results between IF to those achieved using GFP tagged proteins in live cells. It was concluded that protein localization data from fixated cells satisfactory represented the situation in vivo and together exhibit a powerful approach for confirming localizations of yet uncharacterized proteins. Finally, a global analysis based on IF data of approximately 20 % of the human proteome was performed, providing a first overview of the subcellular landscape in three different cell lines. It was found that the intracellular distribution of proteins is complex, with many proteins occurring in several organelles. The results also confirmed the close relationship between protein function and localization, which in a way further strengthens the accuracy of the IF approach for detection of proteins at the subcellular level. / <p>QC 20121017</p> / The Human Protein Atlas

Inom klinisk patologi bedöms vävnadsprover under mikroskop, med syftet att fastställa diagnos. Utgångsmaterial är antingen ett helt organ eller en liten bit från det sjukligt förändrade organet. I det senare fallet kallas preparatet för biopsi och dessa tas med hjälp av biopsiverktyg och oftast under ultraljudledning. Njurbiopsier kan tas vid misstänkt transplantatavstötning, av medicinska skäl, vid inflammatoriska tillstånd (nefriter) samt nefrotiska tillstånd. En del av biopsimaterialet fixeras i 4 % formaldehyd för ljusmikroskopisk (LM) bedömning, medan resten transporteras i transportlösningar som Michels- eller Zeus lösning. Det materialet fryses ned direkt för kryostatsnittning, dessa transportlösningar syftar till att bevara vävnaden färsk för immunofluorescens (IF) analys. Problem kan uppstå när materialet som bedöms med LM inte innehåller ett acceptabelt antal glomeruli, där minst 12- 15 stycken krävs för att biopsin skall vara representativ. Om det är alltför få till antalet brukar man tina och formaldehydfixera IF materialet. Morfologin blir oftast dålig och inte lämplig för bedömning. Den enda som man kan göra är att ombiopsera patienten. Detta är kostsamt, obekvämt för patienten och inte helt riskfritt. Syftet med den här studien var att undersöka ifall man kan ersätta transportlösningarna med ett fixativ som skulle medföra bättre morfologi i de fall som IF materialet måste tinas upp och användas för LM, men samtidigt erbjuda samma kvalitet på IF-signalen. Tre fixeringsmedel jämfördes med rutinmetodens 4 % formaldehyd; dels kommersiellt tillgängliga som Histochoice och HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) och ett fixativ som är baserat på zinkjoner. Resultatet visade att när det gäller LM-undersökning på paraffininbäddat material så gav HOPE och Histochoice acceptabel morfologi av glomeruli. Vid upptinande av materialet följt av formaldehyd/paraffin, sågs fortsatt god kvalitet i den glomerulära morfologin, medan den tubulära morfologin var klart undermålig. Zn-fixering ledde till bubblor i cytoplasman vilket inte är acceptabel morfologi. I IF-analys gav alla fixativ negativ signal utom Zn-fixering, vilken gav en positiv signal. Det innebär att ingen säker diagnos kan ställas vid användning av de undersökta fixeringsmedlen som kan ersätta transportlösningar eller rutinlaboratoriet formaldehyd. Zn-fixativ såg lovande ut om det bortses från bubblorna, men detta behöver undersökas ytterligare i framtida studier / lt is important for clinical pathology to assess tissue samples (preparations) under the microscope to make a diagnosis. Starting materials is either a whole organ, or a small piece of the diseased body. The material can also be in the form of so-called biopsies, which are taken by biopsytools and ultrasound. Kidney biopsy may be taken for suspected transplant rejection, medical reasons and at nephritic (inflammation), and nephrotic states. Half of the material from kidney biopsies are transported in transport solutions of the type Michel / Zeus that serves the purpose of preserving the tissue fresh for immunofluorescence (IF) analysis. The other half is fixed in 4% formaldehyde for light microscope (LM) assessment, a procedure that precludes IF staining due to destruction of the antigenicity of the tissues. Problems might occur when the material is assessed by LM and do not contain an acceptable number of glomeruli, at least 12-15 are regarded as acceptable for diagnosis. If these are fewer in numbers, the material previously used for IF may be thawed and formaldehyde fixed. The morphology is however often adversely affected by this procedure, rendering the tissue unsuitable for morphological assessment. Therefore the only remaining option is to re-biopsy patients. This is costly, inconvenient for the parients, and not completely safe. This study was performed in order to explore if transport solutions can be replaced by a fixative that would result in a better morphology after thawing of the tissues, but with retained IF -quality. Three fixing agents were compared with routine method 4% formaldehyde; the commercially available Histochoice and HOPE fixatives (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) and a fixative that was based on zinc ions. The results showed that in terms of LM, the fixatives HOPE and Histochoice gave acceptable morphology of the glomeruli, while the tubules were poor. It also showed that there were a lot of bubbles in the cytoplasm at the Zn-fixing. As for IF-analysis, all were negative except Zn that gave a positive signal. This means that no secure diagnosis can replace the routine transport formaldehyde. Zn fixative looked promising, except the bubbles, but needs to be further investigated in future studies.

Background During development, endothelial cells acquire mesenchymal-like properties to migrate and facilitate normal vascular formation. This process of transformation is known as endothelial to mesenchymal transition (EndMT) and has also been implicated in diseases like vascular pathologies contributing to endothelial inflammation, atherosclerosis and tumour angiogenesis. The Angiomotin family of scaffold proteins play a role in transducing mechanical force at cell junctions. Of this family, Angiomotin-Like 2 (AmotL2) localises to endothelial cell junctions and was recently found to play a role in regulating endothelial cell mechanosensing and inflammation. Methods/Materials Primary human endothelial cell lines (HUVEC) were cultured and manipulated in vitro to investigate the role of AmotL2 in EndMT. Lentiviral short hairpin RNA interference was employed in AmotL2-loss-of-function studies, (produced using HEK - Human Embryonic Kidney - cells) to generate knockdown(kd) cells. Western blotting (WB) was used to assess AmotL2 depletion and changes in protein expression of key EndMT markers. qPCR was performed to look at the same at a transcriptional level. Immunofluorescent staining and confocal imaging were performed to validate WB and qPCR results as well as to study protein localisation. Results AmotL2 was found to regulate Snail1 and N-cadherin at both protein and mRNA levels. Morphological findings displayed the AmotL2kd cells to be elongated, deviating from the regular cobblestone morphology observed in control cells. An increase in scaffold protein levels was observed in the AmotL2 kd samples. Similar results were seen in qPCR data where increased mRNA expression was observed in the AmotL2 kd samples for the same targets. On analysis of IF image data, more nuclear staining was observed in the kd samples. qPCR analysis done on samples treated with TGF-β, exhibited an increase in mRNA expression of targets involved in the EndMT pathway in the treatment samples against the controls. Conclusion The results suggest that AmotL2 plays a role in EndMT by affecting the transcription factors and proteins involved in the pathway, which leads to changing morphology and behaviour of the cells. Looking into more targets involved in EndMT may give us a better understanding of how this process leads to diseases like atherosclerosis and tumour angiogenesis.