Browsing by discipline "Biokemia"

Lymphangiogenesis is the process that leads to the formation of lymphatic vessels from pre-existing vessels. Vascular endothelial growth factor C (VEGF-C), the ma- jor lymphangiogenic growth factor, is produced as an inactive precursor and needs to be proteolytically processed into a mature form in order to activate its receptors VEGFR-3 and VEGFR-2. A deficiency of VEGF-C during embryonic lymphangiogenesis results in embryonic lethality due to the lack of lymphatic vasculature. Hennekam lymphangiectasia-lymphedema syndrome (OMIM 235510) is in a subset of patients associated with mutations in the collagen- and calcium-binding EGF domains 1 (CCBE1 ) gene. CCBE1 and VEGF-C act at the same stage during embryonic lymphangiogenesis and their deficiency results in similar lymphatic defects. The mechanism behind the lymphatic phenotype caused by CCBE1 mutations is un- known. The aim of this study was to investigate the potential link between VEGF-C and CCBE1 that could contribute to the lymphatic phenotype. In this study, 293T cells were used to observe the effect of CCBE1 on VEGF-C pro- cessing. The co-transfection of constructs coding for CCBE1 and VEGF-C showed processing of the inactive pro-VEGF-C into the active, mature form. However, this processing was efficient only in 293T cells. When CCBE1 from 293T supernatant was purified, A disintegrin and metalloproteinase with thrombospondin type 1 motif 3 (ADAMTS3) co-purified with CCBE1. The levels of pro-VEGF-C and active VEGF-C were monitored by immunoblotting or immunoprecipitating metabolically labeled supernatant with specific antibodies or receptors followed by autoradiography. The activity of the processed VEGF-C was verified by proliferation of Ba/F3 cells stably expressing VEGFR-3/EpoR or VEGFR-2/EpoR chimeras. Furthermore, a VEGFR-3 phosphorylation assay was performed in PAE (Porcine Aortic Endotheial) cells to study details of the CCBE1-mediated regulation of VEGF-C. We found that CCBE1 increases the proteolytic processing of pro-VEGF-C, thereby resulting in increased activity of VEGF-C. CCBE1 itself has no effect on VEGF-C activity but regulates VEGF-C by modulating the activity of the ADAMTS3 protease. We also found that both pro- and mature- VEGF-C can bind to VEGFR-3 but only mature form is able to induce VEGFR-3-mediated signaling. In addition to cleaving VEGF-C, ADAMTS3 was found to directly or indirectly mediate CCBE1 cleavage. The N-terminal amino acid sequence of the ADAMTS3-processed VEGF-C confirmed that ADAMTS3 is the protease responsible for the activation of VEGF-C by 293 cells. Hence, we have identified a mechanism that regulates VEGF-C activity. This mechanism suggests the possible use of CCBE1 as a therapeutic means to treat diseases that involve the lymphatic system.

The International Biology Olympiad is a yearly science competition; hundreds of high school students from over 60 countries take part in it. In Finland, the students are chosen by the national biology competition and the national Olympiad training camp. The research subject of this study is Finnish training for International Biology Olympiad (IBO). About ten high school students who are interested in biology take part at each training camp. Science competitions, such as IBO and training camps, are classified as non-formal or out-of-school science education. Since there is very little scientific knowledge about non-formal biology education, especially about science competitions, the theoretical framework of this study consists of non-formal science education, the relevance of science education and the development of interest. In this study, design research methodology with three research cycles was applied to develop the course. In the study, both theoretical and empirical problem analyses were used. There were two main research questions: 1) What are the needs for the development of the training camp? 2) What kind of training is relevant for the participants? The first research question was elaborated by examining a) what kind of topics of interest the participants have, b) how the participants expect the training to be relevant for them, c) what kind of relevance the previous participants experienced, and d) what kind of effects the previous training camps had on the participants interest in biology and career choice. The features of relevant biology Olympiad training were searched in the second research question. The data was collected from pre-camp and post-camp questionnaires, post-camp interviews and a questionnaire sent for the previous camp participants. The qualitative data was analyzed by content analysis and quantitative data from the questionnaires was analyzed by statistical methods. The main results were following: 1) The biggest needs for development were diversifying the contents and balancing the workload of the camp. The participants of the training camps were interested mostly about medicine and human biology -related topics and cell and molecular biology. In addition, it was found out that the previous participants considered biology education to be individually, vocationally and societally relevant for them. According to them, the training camps were especially individually relevant and had some effects on the career choice. 2) The new training camp for biology Olympiad is especially individually relevant for the participants but it has also some vocational relevance. In the science Olympiad training, special attention should be directed towards developing vocational and societal relevance. A new model for Biology Olympiad training camps was developed based on the collected research data. The individual dimension can be improved by i) diversifying the contents of the camps, ii) including inquiry-based learning modules and by iii) taking a student-centered approach to the development process. The vocational and societal dimensions of relevance can be targeted by iv) allowing the teachers of the participants to take part at camps. The vocational dimension of relevance can be enhanced v) by organizing visits to research laboratories and companies and vi) by enabling the participants to meet university students. The results of this study can be applied to not only to Science Olympiad training but also to non-formal biology education. This research provides models for developing out-of-school biology education and its relevance. This is the first design research study about Biology Olympiad training and it opens up discussion about the relevance of science competitions and Science Olympiad training.

Autophagy is a eukaryotic cellular process where intracellular material is recycled by transporting it in newly formed vesicles to lysosomes for degradation. In normal conditions autophagy supports cellular homeostasis. Different stress conditions can induce autophagy and then it helps the cell to avoid an unnecessary or uncontrolled cell death. RAB proteins are small GTPases that regulate vesicle traffic and fusion events in endocytic and exocytic pathways. RAB24 has recently been shown to participate in autophagy, but there is very little information about how it works at the molecular level. GOSR1 is a Golgi SNARE protein that regulates membrane fusion events, and it has been observed to interact indirectly with RAB24. The participation of GOSR1 in autophagy has not been studied yet. The aim of the study was to find out if RAB24 and GOSR1 colocalize into the same vesicle structures and if they interact within each other. HeLa cells were used as a model organism, and to induce autophagy amino acid starvation was used. For GOSR1 detection a DNA construct was created where GOSR1 was tagged with green fluorescent protein GFP-sequence. Localization was studied with immunofluorescence staining where in addition to RAB24 and GOSR1 also the autophagosomal marker protein LC3 was labeled. The labeled cells were photographed with a confocal microscope. The pictures were analyzed with ImagePro software. Interaction between the proteins was studied using immunoprecipitation. GOSR1 and RAB24 were not observed to colocalize into same structures in significant amount. Instead it was found that GOSR1 colocalized into LC3-positive autophagic vesicles. In immunoprecipitation studies no interaction between RAB24 and GOSR1 could be shown. In order to ensure the results more immunofluorescence stainings should be done using several time points and GFP-tagged GOSR1. Also GOSR1 silencing with siRNA should be used in order to find out if GOSR1 is necessary for autophagy. The immunoprecipitation protocol should be optimized, and the possible interaction could be studied by using other methods, for example yeast-two hybride technology.

Microvesicles (MVs) are lipid bilayered membranous vesicles containing functional lipids, proteins, RNA and DNA that are produced by most cells. The physiological significance of MVs has become evident, and increased MV counts and the contents of MVs are nowadays also associated with different pathophysiological phenomena. The goal of the field is to use MVs as diagnostic and therapeutic tools. To achieve this, the understanding of the mechanisms of the functions of MVs should be understood better and additionally, reliable methods for the quantification and characterization of MVs should be developed and standardized. The aim of the study was to determine differences in platelet-derived MVs produced by different activation mechanisms. The second aim was to set up and optimize a protocol based on the reaction of sulphur, phosphate and vanillin (SPV) for measuring lipid content of MVs. The third aim was to study the effect of thrombin and proteinase inhibitor PPACK to the vesiculation of platelets. Platelets were isolated from the whole blood of healthy volunteers and vesicles were produced by platelet agonists mediating thrombogenic activation (thrombin and collagen, TC), pathophysiological activation (lipopolysaccharide, LPS) and Ca-ionophore (A23187) as positive control for vesiculation. Quantification and size determination of produced MVs was done using Nanoparticle Tracking Analysis (NTA). MVs were characterized by protein content using bicinchonic acid assay (BCA) and by lipid content using SPV-reaction. MVs had great activation-dependent differences in the lipid and the protein content. Activation with Ca-ionophore produced the most MVs, but the lipid and protein content was only a fraction from (patho)physiologically induced MVs. Only TC increased vesiculation. Vesicle subpopulations had significant difference in lipid content. Thrombin and proteinase inhibitor PPACK mediated inhibition of platelet formation in all of the activations, but the effect was not statistically significant. The mechanism of inhibition was likely to be proteinase inhibitor mediated. The isolation of vesicle populations using differential centrifugation proved to isolate studied populations only partially and the quantification method with NTA was susceptible to concentrated samples. SPV protocol reacted with different intensity to different lipids. In the future, quantification and isolation methods for MVs and the subpopulations of MVs should be improved. Additionally, to understand the physiologically relevant mechanisms of platelet-derived vesicle formation, the inhibitor experiments with PPACK should be continued, because the number of replicates was too low to see significant effects due to a large donor-dependent deviation. Since MVs are heterogenous cellular multitools affecting varying (patho)physiological phenomena, optimization and standardization of methods should be continued in order to study MVs properly.