Supervisors info:
Σίδερης Διαμάντης, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ, (Επιβλέπων)
Σκορίλας Ανδρέας, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ,
Αυγέρης Μαργαρίτης, Αναπληρωτής Καθηγητής, Τμήμα Ιατρικής, ΕΚΠΑ
Summary:
Epilepsy is defined as the disease characterized by a predisposition of the brain to produce epileptic seizures. An epileptic seizure is a paroxysmal event characterized by a series of repetitive electrical discharges of a pathologically synchronized population of neurons in the cerebral cortex and is manifested by a sudden onset of disturbance of consciousness and/or various mental and physical functions (eg loss of bladder and bowel control). According to epidemiological data, epilepsy affects approximately 50 million people worldwide, and moreover, it often appears in childhood. Epilepsies are classified based on their etiology while at the same time, according to the location of the crises in the brain. As a first line of defense in the therapeutic course, patients are given so-called antiepileptic drugs (AEDs). However, despite the availability of more than 20 AEDs, a remarkable percentage of 33% suffer from drug resistance and therefore seizures are uncontrollable and do not stop regardless of AED intake. Therefore, it is necessary to treat it with alternative therapeutic approaches. tRNA fragments (tRFs) are a group of small non-coding RNA molecules derived from transfer RNAs (tRNAs). The production of tRFs has been observed in several diseases such as cancer, various neurological and infectious diseases and epilepsy. Studies have revealed that specific tRNA fragments detected in the plasma of epileptic patients are associated with epilepsy. In this thesis, optimized methods for the detection and quantification of tRFs were applied in two groups of epileptic patients: those who show resistance to medication and those who respond to it. The studied molecules are tRF derived from lysine tRNA 5΄-tRF-LysCTT, tRF derived from phenylalanine tRNA 3'-tRF-PheGAA, tRF derived from glutamic acid tRNA 5'tRF -Glu-CTC and the tRF derived from glycine tRNA 5'-tRF-GlyGCC. For this purpose, peripheral blood serum samples were collected and analyzed from the two groups of epileptic patients. Specifically, 72 serum samples from patients with epilepsy were used, of which 37 came from patients who
responded to medication and 35 from patients who did not respond to medication. The exogenous reference miRNA cel-miR-39 of the worm C. elegans was used to normalize the results between the different samples. In addition, RNA sample from the HeLa cell line was used as a calibrator to evaluate the results of the PCR reactions. The experimental procedures applied include the isolation of total RNA, the polyadenylation reaction, the reverse transcription reaction of the RNA into cDNA and finally the real-time quantitative PCR reaction using the dye SYBR Green I. Based on the results of the levels of the studied circulating tRFs in the samples, an extensive biostatistical analysis was performed to evaluate the differential diagnostic and prognostic clinical value of these molecules for the patients with drug-resistant epilepsy. Initially, in order to distinguish the patients who will show resistance to medication, an analysis was carried out through the construction of ROC (Receive Operator Characteristic Curves) sensitivity and specificity curves. The analysis shows that there are increased levels of the tRFs under study in the serum of the non-responsive group compared to the responsive group. In addition, to represent the distribution of the levels of tRFs in the patients' serum, boxplots were constructed in which it is confirmed that there is a trend of increased levels of tRFs in the non-responsive group. Following, a spearman correlation analysis was performed, through which the importance of the correlation of the levels of circulating tRFs in the serum of patients with epilepsy is highlighted. Finally, univariate logistic regression models were constructed. Through these models, the relative risk was calculated for the tRFs and their independent prognostic value as indicators of drug-resistant epilepsy was estimated. From this analysis it appears that patients who have elevated levels of the studied tRFs have a greater risk of developing drug resistance.
Keywords:
epilepsy, drug resistant, tRFs, PCR
