Post-graduate theses
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| Identifier |
000392817 |
| Title |
hGDH1 and 2 over-expression and co-expression in cell lines |
| Alternative Title |
hGDH1 και 2 υπερέκφραση και συν-έκφραση σε κυτταρικές σειρές |
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Author
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Μαθιουδάκης, Λάμπρος
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Thesis advisor
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Ζαγανάς, Ιωάννης
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| Abstract |
Background: Glutamate dehydrogenases catalyze the reversible deamination of L-glutamate to a-ketoglutarate and ammonia. Two isoforms of glutamate dehydrogenases are found in humans, namely hGDH1 and hGDH2. hGDH1 is expressed in almost every human cell (housekeeping enzyme). On the other hand, hGDH2 has been found mainly in brain, testis and kidneys. Both enzymes are allosterically activated by ADP and L-leucine and inhibited by GTP. They contribute to important cellular processes as they interconnect amino acid and carbohydrate metabolism, mediate neurotransmitter recycling and play important role in energy production, ammonia management and insulin secretion. Previous studies have indirectly proven that both enzymes are located in mitochondria matrix.
Aim of the study: The present study is divided in two main complementary objectives. The first objective is to determine whether the two isoenzymes are co-localized subcellularly. The second objective is to study the result of the over expression of hGDH1 and hGDH2 in mammalian cell lines.
Methods: For answering our first scientific question, we tagged each iso-enzyme with fluorescent protein of different colour, so that we could detect them subcellularly using confocal microscopy. For the second objective, we created stable HEK293 cell lines over expressing either hGDH1 or hGDH2 in order to study their phenotypic characteristics.
Results: Experiments related to subcellular localization, revealed that the two enzymes are co-localized inside mitochondrial matrix. Specifically, after tagging hGDH1 with GFP and hGDH2 with RFP, it was confirmed, as far as the resolution of our confocal microscope permitted, that they co-localize inside the mitochondrial matrix. Moreover, characterization experiments showed that stable HEK293 lines over expressing hGDH1 or hGDH2, respectively, display lower death rates in different time points of their lifespan as compared to wild-type cells. Specifically, mainly in exponential growth phase (24h after plating), WT HEK293 exhibit more dead cells (approximately 35%) compared to stable cell lines over expressing hGDH1 and hGDH2 (10-15%; p<0.0001 and 5-15%; p <0.0001, respectively).
Conclusions: In conclusion, our experimental results suggest subcellular co-localization of hGDH1 and hGDH2, as well as major contribution of both iso-enzymes in cell growth and lifespan. These data need to be further analyzed and followed by additional studies for deciphering the role of hGDHs in cell viability and function.
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| Language |
English |
| Issue date |
2015-03-31 |
| Collection
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School/Department--School of Medicine--Department of Medicine--Post-graduate theses
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Type of Work--Post-graduate theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/5/a/f/metadata-dlib-1431673125-604227-16649.tkl
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| Views |
209 |
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