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Department of Medical Cell Biology, Uppsala

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Abstract bachelor project 2017-2018: The involvement of endoplasmic reticulum - mitochondrial contacts in functional and dysfunctional beta cells

Mitochondrial-endoplasmic reticulum contacts are connections between mitochondria and the endoplasmic reticulum that play a key role in the regulation of lipid synthesis, Ca2+ signaling, intracellular trafficking and the control of mitochondrial biogenesis. Ca2+ signaling influences insulin secretion in beta cells so it is possible that these contacts might have an involvement in dysfunctional beta cells like diabetic beta cells. Moreover, it has been shown that diabetic humans have a decreased amount of these contacts. The role these contacts have in insulin secretion is not known. This project aims to investigate the effects of increased contacts by recruiting the mitochondria to the endoplasmic reticulum using the optogenetic Cry2-CIBN technique that can induce dimerization of two proteins in blue light, resulting in more of these contacts.

Calcium signaling influences insulin secretion among other things. Calcium efflux from the endoplasmic reticulum as well as influx from the extracellular medium gets triggered when the plasma membrane of the cell is depolarized. A spiking pattern can be seen because the concentration of calcium changes rapidly. The calcium signaling is studied using total internal reflection fluorescence microscopy by transfecting INS1 cells with optogenetic constructs and a red genetically encoded calcium indicator (RGECO). A 30mM potassium buffer is used to depolarize the plasma membrane and thus induce calcium signaling. The effect of the mitochondria-endoplasmic reticulum induced contacts can be studied by comparing its spiking pattern to a control without active recruitment of mitochondria to the ER. This project shows a decrease in the number of spikes but an increase in the height of these spikes when mitochondria are recruited to the endoplasmic reticulum.

The insulin secretion of INS1 cells was also studied. This was done by transfecting the cells with optogenetic constructs. Samples were taken from a basal buffer and potassium buffer which had incubated for thirty minutes. These samples were than tested using an AlphaLISA to determine the insulin in the sample. Transfected and untransfected cells were compared as well as cells incubated in blue light and in darkness. This project shows a tendency for higher insulin secretion when mitochondria are recruited to the endoplasmic reticulum when compared to cells where the mitochondria are not recruited to the ER.

This study shows that the amount of mitochondria-endoplasmic reticulum contacts does affect the calcium signaling and the insulin secretion. Moreover, it shows that an increase in these contacts causes a decrease in calcium signaling spikes but an increase in its heights and an increase in insulin secretion. The effect of these contacts when they are reduced should be tested and if the opposite is true and a decrease in these contacts as seen in diabetes really prove to be of a lowering effect on the insulin secretion, new drugs might be able to be developed to increase the number of contacts in diabetic patient, restoring the insulin secretion to a normal or near normal level

Abstract bachelorproef 2016-2017: The effect of 4μ8C on hepatic stellate cells and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver. It is a multistage disease linked to environmental, dietary and lifestyle factors. Most cases arise in patients with an underlying liver disease like cirrhosis. Hepatic stellate cells play a key role in development of cirrhosis and in the progression of hepatocellular carcinoma. Interplay between hepatic stellate cells and tumor cell is bidirectional by mutual promotion of each other’s proliferation and differentiation. Therefore, it has been proposed that blocking hepatic stellate cell activation could be used as a promising therapy for patients with HCC. The specific IRE1a inhibitor 4µ8C has been shown to block activation of stellate cells and fibroblasts in vivo and in vitro.

This current study now focusses on the effects of 4µ8C on hepatic stellate cell activation in the context of liver cancer and on interactions between hepatic stellate cells and tumor cells. The methods that were used are: a chemically induced mouse model, which gives a good representation of HCC; a transwell assay, to study the interactions between HSC and tumor cells; QPCR analysis, to measure certain markers that are linked with ER stress and an alamar blue assay to determine cell proliferation and viability.

The results indicate the tumor cells secrete factors that induce ER-stress in hepatic stellate cells. We also show that blocking the IRE1a ER-stress pathway with the specific inhibitor 4u8C decreases tumor burden in the mouse model for HCC and that 4u8C decreases migration and proliferation in tumor-stellate cell co-cultures. Further research is necessary to clarify the role of ER-stress in the cross-talk between stellate cells and tumor cells.

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Husargatan 3
751 23 Uppsala
Sweden

Contacts

Femke Heindryckx
femke.heindryckx@mcb.uu.se
Traineeship supervisor
Nadine Griesche
Nadine.Griesche@mcb.uu.se
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