Pirkanmaa University of Applied Sciences (PIRAMK) Tampere
Knowledge, skills and competence to be acquired
Basic knowledge of mitochondrial research, in particular mitochondrial metabolism. Basic knowledge of the study of post-translational protein modification. Knowledge, skills and competence in the various practical procedures to be used during the training period, as follows:
Culture and maintenance of various cell lines. Cell transfection. Isolation of cultured cells and preparation of fractions for protein analysis by Western blotting. Measurement of various parameters of metabolism, mostly using the Seahorse XF24 instrument (measurement of oxygen consumption and medium acidification), of cells undergoing various metabolic treatments. Critical evaluation, analysis and correct presentation of results.
Detailed programme of the training period
Post-translational modifications of proteins have important regulatory functions. One such modification is the reversible acetylation of lysine residues. We study the function of three highly conserved protein deacetylases of the Siruin protein family that function in the mitochondrial compartment. To this end we have established cell lines that can be induced to express these deacetylases. Alternatively their expression can be inhibited (knock-down) using RNA interference. During the training period we will test effects of overexpression or knockdown as well as various pharmaceutical reagents that interfere with Siruin function on mitochondrial metabolism in cultured cells and at the same time measure mitochondrial protein acetylation and other parameters of mitochondrial function.
Tasks of the trainee
The proposed research is partly self-contained and requires the trainee to both plan and execute the project in coordination with supervisor(s). The trainee will during the project keep a detailed labbook, present and discuss results at regular labmeetings and meetings with supervisor(s) and eventually write a report.
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Samenvatting eindwerk 2014-2015: Analyzing mutant zebrafish lines in their resistance against Mycobacterium marinum
Tuberculosis remains a worldwide health problem caused by Mycobacterium tuberculosis (M. tuberculosis). The pulmonary tuberculosis caused by this bacterium is very contagious. Therefore, an improved treatments are needed for tuberculosis. The zebrafish is considered as a good model for mycobacterial pathogenesis since the treatment with Mycobacterium marinum (M. marinum) a natural fish pathogen, causes a systemic chronical infection in zebrafish at 28°C.
It is crucial to get novel information about genetic mechanisms underlying immunological defense against tuberculosis. In this project those mechanisms were studied by using M. marinum infection in zebrafish as a model for tuberculosis. To find out which genes have a role in the immunological defense against M. marinum, zebrafish lines with random mutations were produced. Seven different mutant lines were chosen to be infected and their survival followed because they looked more susceptible for pneumococcal infections in embryonic stage than the wild type AB zebrafish embryos. Each line had both homozygous and heterozygous fish for the mutation. These lines were infected with M. marinum and this way mutant fish that are more susceptible to M. marinum than the negative control wild type AB could be found. These lines can then be used in further research. The embryos were injected with approximately 40 cfu of M. marinum under a microscope into the yolk sac. Then the survival was followed for seven days.
Infections for each line were repeated three times to confirm the results. A potential error source was that the bacterial dose may vary between experiments and also in the same experiment between the groups. The dead rate is linked to the dose so that a higher dose leads to a higher dead rate. One line is not reliable because each experiment shows a different outcome. The outcome of four of the seven lines are similar to the control line AB and can be used as controls. There are two interesting mutant families that appear to react the same as in the pneumococcal infections in which these lines looked more susceptible. These are F1 206 and F1 305.
This makes mutant lines F4 (F1 206) and F4 (F1 305) promising for further research on the innate immunity mechanisms of zebrafish against mycobacterial infection. However, more repeats of the experiments are still needed because of the variation between experiments. The more susceptible lines than the control line AB, such as (F1 206) and F4 (F1 305) can provide novel information when the mutated genes are identified. This information can be useful when developing new treatments for tuberculosis.
Samenvatting eindwerk 2011-2012: Influence of the storage time before centrifugation on blood ionized calcium using a blood gas meter
In Finland, the laboratories are centralized, which means that samples often have to travel some time before they arrive in the laboratory. In addition, not all the samples are already centrifuged. In this study, we try to find an answer on the question if there is a difference in the concentration of ionized calcium between samples which are centrifuged within two hours after the sampling and samples for which the time before centrifugation is longer as normal.
Calcium can be divided in three different fractions, namely protein bounded calcium, complexed calcium and ionized calcium. Ionized calcium (Ca2+) is the divalent ion of calcium. The normal range of ionized calcium concentration in healthy adults is between 1,20 mmol/L and 1,35 mmol/L. When the values are increased, it is been called hypercalcemia, when it is been called hypocalcemia, the values are decreased. The main reason of hypercalcemia is primary hyperparathyroidism, for hypocalcemia, it is hypoparathyroidism.
Five samples of twenty volunteers were collected. The first samples were centrifuged between a half and one hour after sampling, the second sample from each volunteer after two hours and so on until the fifth sample that was centrifuged after five hours. Immediately after the centrifugation, the ionized calcium concentration was measured by the blood gas meter ABL800 FLEX. The pH is measured by an ion-selective electrode with a glass membrane. The concentration of ionized calcium is detected by an ion-selective electrode with a polyvinyl chloride membrane. Both electrodes have the same kind of reference electrode, a silver/silver chloride (Ag+/AgCl) reference electrode.
The results of the samples which are centrifuged after one hour are the results which are considered the real results. First the pH is measured. When the time before the centrifugation increases, the pH-values decreases following a negative exponential function. To investigate whether the values are significantly different the Mann-Whitney and Kruskal-Wallis test were performed. These tests investigate respectively if two or more groups of values are significantly different from each other. For the pH-measurement, after already two hours after centrifugation, the results are significantly different. The results of the measurement of the ionized calcium concentration are the same whether the samples are centrifuged one hour after sampling or whether it takes five hours. The blood gas meter calculates the concentration of ionized calcium at a pH of 7,4. Therefore both earlier results are used and the values of the concentration of ionized calcium corrected for pH value decline when the time before centrifugation increases. As is the pH-measurement, the results follow a negative exponential function. These results are significantly different if it takes longer than two hours before the samples are centrifuged.
If only the concentration of ionized calcium is taken into account, there is no influence of the time before the samples are centrifuged. On the other hand, there is an influence on the pH-measurement and on the measurement of the concentration of ionized calcium at a pH of 7,4. When only the ionized calcium concentration is needed, it does not matter if the sample is not centrifuged. When the results of this concentration are needed at pH 7,4 the samples need to be centrifuged before two hours after sampling.
Samenvatting eindwerk 2008-2009: Methods set up for studying mitochondrial cell metabolism
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