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    Open Access
    Ηθικοδεοντολογικά και νομικά προβλήματα στην υποβοηθούμενη αναπαραγωγή
    (2023-06-08) Δρέττας, Πέτρος; Drettas, Petros
    Ο προεμφυτευτικός γενετικός έλεγχος αποτελεί ένα νέο επιστημονικό εργαλείο ικανό να ανιχνεύει καταστάσεις που μπορούν να αντιμετωπιστούν αμέσως μετά τη γέννηση ή κατά τη διάρκεια της εγκυμοσύνης. Η εφαρμογή του θα μπορούσε να επηρεάσει τόσο την προγεννητική περίοδο όσο και τις τεχνικές υποβοηθούμενης αναπαραγωγής. Είναι πολύ ωφέλιμο καθώς παρέχει πολλές χρήσιμες πληροφορίες στους μελλοντικούς γονείς σχετικά με την ιατρική κατάσταση των απογόνων τους. Επιπλέον, ο ορισμός της «σοβαρής νόσου», που ρυθμίζει την προεμφυτευτική διάγνωση, τη σπερματέγχυση από δότη, ακόμη και τους ορισμούς των προϋποθέσεων για τις ασθένειες της άμβλωσης, θα πρέπει να αναθεωρηθεί, συμπεριλαμβανομένων όλων των κλινικά σοβαρών ασθενειών. Από την άλλη πλευρά, μπορεί να προκύψουν αντιπαραθέσεις ειδικά σχετικά με τη δωρεά γαμετών. Οι μελλοντικοί γονείς και οι απόγονοι ενδέχεται να ενημερωθούν σχετικά με τα δημογραφικά και ιατρικά χαρακτηριστικά των δοτών. Αυτή η μελέτη στοχεύει στη διερεύνηση των επιπτώσεων της εφαρμογής του προεμφυτευτικού γενετικού ελέγχου στην αναμόρφωση των ηθικών κριτηρίων στην υποβοηθούμενη αναπαραγωγή.
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    Open Access
    Ο ρόλος του ηλεκτροεγκεφαλογραφήματος (ΗΕΓ) στην μελέτη σηπτικών ασθενών με και χωρίς συμμετοχή του κεντρικού νευρικού συστήματος. Συσχέτιση ΗΕΓ ευρημάτων με φλεγμονώδεις βιοδείκτες πλάσματος και κλινική έκβαση
    (2023-04-07) Πλατανάκη, Χριστίνα; Platanaki, Christina
    Το Σύνδρομο της Σήψης αποτελεί την τελική έκφραση των λοιμώξεων ανεξαρτήτως αιτιολογίας. Ο ασθενής που βρίσκεται σε σήψη μπορεί να παρουσιάσει διαταραχή του επιπέδου συνείδησης. Σχετικά με τη διάγνωση το ηλεκτροεγκεφαλογράφημα αποτελεί εξαιρετικά ευαίσθητη μέθοδο και μπορεί να αναδείξει βλάβη ακόμα και όταν η φυσική νευρολογική εξέταση είναι φυσιολογική. Στο Γενικό μέρος της παρούσας μελέτης αναλύονται εκτενώς οι έννοιες της Σήψης, της Εγκεφαλοπάθειας σχετιζόμενης με την Σήψη και του ηλεκτροεγκεφαλογραφήματος ως μεθόδου διάγνωσης σε διάφορες καταστάσεις. Στο ειδικό μέρος επιχειρήθηκε αρχικά η ανάδειξη της χρησιμότητας του ηλεκτροεγκεφαλογραφήματος στην κλινική πράξη σε σηπτικούς ασθενείς που νοσηλεύονται σε κλινική εκτός ΜΕΘ. Για τον σκοπό αυτόν πραγματοποιήθηκε βιβλιογραφική ανασκόπηση της πρόσφατης διεθνούς βιβλιογραφίας. Επιπλέον, επιχειρήθηκε η συσχέτιση φλεγμονωδών βιοδεικτών πλάσματος αλλά και του φλεγμονώδους βιοδείκτη ΕΝΥ NGAL με τις ηλεκτροεγκεφαλογραφικές διαταραχές ασθενών με εικόνα εγκεφαλοπάθειας από σήψη χωρίς απαραίτητα πρωτογενή λοίμωξη ΚΝΣ.
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    Open Access
    Identification of synthetic lethal interactions with DNA replication licensing aberrations
    (2023-06) Badra Fajardo, Nibal; Badra Fajardo, Nibal
    Precise and complete genome duplication is critical for a dividing somatic cell to ensure the inheritance of an accurate copy of parental DNA by the offspring. However, multiple DNA barriers commonly arise and disrupt the regular progression of replisomes along replication, posing a threat to timely completion of DNA synthesis within a single cell cycle. DNA licensing controls replication to ensure complete genome duplication while restricting single origin firing to once-per-cell cycle. The CDT1 licensing factor is particularly relevant during licensing due to its critical function in recruiting an inactive form of the replicative MCM2-7 helicase on DNA origins. In order to prevent illegitimate origin firing, licensing is restricted upon origin initiation and helicase unwinding of DNA through tight regulation of each licensing component. It has been demonstrated that CDT1 undergoes multiple control mechanisms in mammalians, underlining its critical relevance when promoting DNA licensing. Accordingly, overexpression of CDT1 is induces genomic instability and malignant behavior in vitro and in vivo and is a genomic trait of several tumor types. One of the mechanisms controlling CDT1 activity involves Geminin, a cell-cycle regulated protein that operates after helicase unwinding of DNA at the onset of S phase until G2, to bind and inhibit CDT1. Certain studies proposed that Geminin could function as a backup pathway to limit CDT1 activity in highly proliferative cells where CDT1 activity is found upregulated. The potential dependency of highly proliferative cells to Geminin could be a genomic trait of therapeutic significance towards the identification of new therapeutic approaches in cancer treatment. However, the molecular mechanisms leading to malignant transformation of cells presenting licensing aberrations was still far from being elucidated. During the initial steps of this study, we validated that CDT1 over-expression leads to re-replication and DNA damage in human cells, and that it is a genomic hallmark of multiple tumor types associated with poor survival. We then focused on the licensing inhibitor Geminin, and we demonstrated its activity is essential to prevent origin re-licensing and re-replication selectively in cancer cells. Cancer cells depleted of Geminin exhibited more than two copies of DNA, evident from cell cycle analysis and increased DNA damage when compared to normal cells. Moreover, we showed that silencing of Geminin triggers replication stress even from the first round of replication, impairing fork progression and 1 replication completion at late-replicating loci. Single-strand DNA gaps were observed to accumulate after progressive rounds of re-replication in Geminin-deficient cancer cells, suggesting that Geminin inactivation triggers deregulated origin initiation. Consistent with this, activation of the DNA damage G2/M checkpoint was required for re- replication, whereby inhibiting checkpoint signaling led to mitotic catastrophe and increased genomic instability during the next cell cycle. Given that deregulated licensing is a genomic signature of cancer, we then sought to identify targetable molecular pathways signaling and responding to this phenomenon by performing a synthetic-lethal high content screening in cell models with aberrant licensing by examining proliferation and DNA damage. After validation of primary hits, we demonstrated the central effector of the Fanconi anemia repair pathway FANCD2 as critical during protection of cells with induced re-replication. Geminin and FANCD2 double-depletes exhibited reduced survival and increased DNA damage and these results were also confirmed in patient-derived FANCD2-KO cells depleted of Geminin. Remarkably, we observed that Geminin absence induces FA signaling early during the first round of replication, as demonstrated by immunostaining and chromatin fractionation assays. FANCD2 foci were partially distinct from DSBs, indicating sites of impaired fork progression followed by DNA breakage. Subsequent analyses revealed that cells undergoing re-replication require both canonical and non-canonical FANCD2 activity to restrict extensive genomic instability; Loss of FANCD2 in Geminin-depleted cells induced deregulated origin initiation and increased replisome progression, triggering in turn replication through single-strand DNA templates, fork collapse and chromosome fragility. The results in this study provide a novel synthetic lethal interaction with therapeutic relevance in targeted-studies, by saturating the DNA repair capacity of cancer cells presenting licensing aberrations for selective cancer-cell killing.
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    Open Access
    Patient radiation dose in the 2-phase computed tomography imaging of the parathyroid glands
    (2023-05-12) Δημητρούκας, Χρήστος; Dimitroukas, Christos
    A large dose audit was initially performed to evaluate the radiation dose to 573 adult patients, who underwent eleven common CT examinations in two different scanners: one with 16 detector rows (GE Light Speed) and one with 80 detector rows (Toshiba Aquilion Prime). The CTDIvol, DLP, as well as patients’ demographic and technical data were recorded. The 75th percentiles of the distribution of the CTDIvol and DLP values were proposed as IDRLs and were compared to corresponding NDRLs and international values. All dose values are based on the 32-cm body phantom, except for the head, sinuses and auditory canal examinations in Toshiba system, as well as the head and neck examinations in GE system where the 16-cm head phantom is used. A reduction of up to 67% and 64% (Mann-Whitney test p<0.05) was reported for the mean CTDIvol and DLP values obtained with the Toshiba system compared to the GE system. The IDRLs for the Toshiba system were lower up to 71% and 68% compared to the corresponding NDRLs in terms of CTDIvol and DLP values, respectively. For the GE system, the IDRLs regarding the CTDIvol values were lower up to 23% and 39% compared to the corresponding NDRLs, except for the skull base examination that exceeded up to 112%, as well as for the head and lumbar spine examinations that were exceeded 10%, regarding the DLP values. The ED was also calculated based on the recorded DLP values and suitable conversion coefficients. Focusing on the neck region, the mean ED values were 2.95 mSv for the GE system and 2.02 mSv for the Toshiba system. These values were lower than those reported in most of the international studies previously published. A following study aimed to evaluate the radiation dose during five CTA examinations (brain, carotids, thoracic aorta, abdomen aorta, pulmonary arteries) performed with the above CT systems, and to establish IDRLs. The CTDIvol, DLP per examination, patients’ demographic and anatomical data, and technical data were retrospectively recorded for 240 adult patients. The 75th percentiles of the distribution of the CTDIvol and DLP values were again proposed as IDRLs. The radiation dose values were compared to corresponding previously published international data. Focusing on the neck region (carotids), the IDRLs were 6.5 mGy and 280 mGycm, in terms of CTDIvol and DLP values, respectively, for the Toshiba system. The respective mean ED value was 2.5 mSv. The reductions of the obtained CTDIvol and DLP values obtained with the Toshiba compared to the GE system were associated with the implementation of dose-saving technologies, such as lowered tube voltage and IR algorithm. The Toshiba system’s IDRLs and ED values were comparable or lower than those reported in most of the previously published studies. There are significant variations in patient doses during CTA examinations, due to the CT systems’ differences, scanning parameters and departmental CTA protocols utilised. These two introductory studies were focused on the neck region, i.e. standard neck examinations (one phase) and carotids’ CTAs (two phases), respectively. Patient dose surveys could contribute towards optimising radiation protection for patients; therefore, highlighting the necessity to increase the awareness and knowledge of the radiation dose levels during common CT or specific CTA examinations. The patient radiation dose from the two-phase parathyroid protocols of the two different CT systems applied in our Hospital was then evaluated and compared with that delivered by the other similar protocols previously published. 214 patients with PHPT were included in the study with a two-phase CT scan by using the above two scanners. The standard ‘neck’ or a modified ‘parathyroid’ protocol was used. The patient dose was again evaluated in terms of CTDIvol, DLP and ED per acquisition protocol and CT system. CTDIvol and DLP were recorded retrospectively, while the ED was calculated based on DLP and an appropriate conversion coefficient. Comparisons of patient dose between the two protocols and two CT systems and the corresponding published values were established. A significantly lower patient dose (40.2–43.2%) than the GE system (p<0.0001) resulted from the Toshiba system. The ‘parathyroid’ protocol resulted in a 6.5–9.6% lower patient dose than the standard ‘neck’ protocol. Compared with the literature, the lowest ED value (3.6 mSv) was observed since this protocol consists of a lowered tube voltage of 100 kVp, a reduced scan length for the pre-contrast phase and implementation of an IR algorithm. The objective of the next study was to estimate organs’ absorbed dose from the two-phase CT of parathyroid glands and ED based on three different methods, and to compare the dose values with those reported by other published protocols. CTDIvol, DLP, and the corresponding scan length during each phase of the modified parathyroid protocol were recorded, for 76 patients. One k-factor, and two different k-factors for the neck and chest area were used to estimate the ED from DLP. A Monte Carlo software, VirtualDoseCT, was also used for the estimation of organs’ absorbed dose and ED. Two-phase parathyroid CT resulted in a mean ED of 3.93 mSv, 4.29 mSv and 4.21 mSv according to the one k-factor, two k-factors, and VirtualDoseCT methods, respectively. The two k-factors method resulted in a slight overestimation of 1.9% in total ED compared to VirtualDoseCT. No statistically significant difference was found in ED values between these methods (Wilcoxon test, p>0.05), except for female patients in the pre-contrast phase. The organs inside the SFOV received the following doses: thymus 23.3 mGy, lungs 11.5 mGy, oesophagus 9.2 mGy, thyroid 6.9 mGy, and breast 6.3 mGy. The ED and organs’ dose were significantly lower in the pre-contrast than in the arterial phase (Wilcoxon test, p<0.001). A statistically significant difference was observed between male and female patients for the pre-contrast phase (Mann-Whitney test, p<0.05), regarding the ED values obtained with the two k-factors method and VirtualDoseCT software. The two k-factors method could be considered as safe for the ED estimation in clinical practice, if appropriate software is not available. A wide range of ED values was found in the literature, mainly depending on the acquisition protocol parameters and the estimation method. At our Hospital, the ED is reduced by decreasing the tube voltage, scan length and number of phases. Another study aimed to assess the influence of overranging on patient ED and radiosensitive organs' dose (OD) of adult patients undergoing two-phase parathyroid CT examinations utilising Monte Carlo simulation and propose methods for elimination of its impact in clinical practice. The CTDIvol, and DLP per examination, demographic and anatomical data, as well as technical data were retrospectively recorded for seventy-six patients. The ED and OD values were calculated for both sexes, and phases and as a total, with and without considering overranging, using VirtualDoseCT software incorporating a Toshiba Aquilion Prime 80 CT scanner and the recorded parameters. A significant reduction of about 30% (Wilcoxon test p<0.0001) was found in total ED values obtained without considering overranging (3.2 vs 4.2 mSv). A significant decrease was also found in total ODs without considering overranging for females (except for the thyroid), males and patients’ total sample (Wilcoxon test, p<0.05). The percentage differences in total ODs between the two methods were 2.1% for thyroid, 14.1% for oesophagus, 14.6% for thymus, 19.0% for eye lenses, 26.3% for lungs, and 83.5% for breasts. A non-significant decrease of 4.6% (Mann-Whitney test, p=0.259) was found in total ED values obtained without considering overranging for males compared to females (3.12 vs 3.26 mSv). In addition, a significant reduction was found in total ODs for males compared to females for thyroid, oesophagus, and eye lenses (Mann-Whitney test, p<0.0001), but not for breasts (Mann-Whitney test, p=0.061), and a non-significant increase for the lungs and thymus (Mann-Whitney test, p>0.05). Our results highlight that overranging should be of particular concern when optimisation strategies are planned during parathyroid CT imaging, since there is the potential to significantly reduce ED and OD absorbed by radiosensitive organs inside or adjacent to the scanning volume. Further work needs to be conducted, combining physical and technical parameters affecting overranging, to increase the awareness and understanding of the unnecessary patient dose during CT imaging, and motivate CT facilities to implement dose optimisation strategies. The purpose of the final study was to review the published literature and compare the different MDCT protocols used for PGs’ imaging. This was accomplished in terms of clinical and technical parameters of the applied protocols, the diagnostic efficacy and patient dose. An extended discussion was performed regarding the discrepancy in the appropriate combination of phases, clinical and technical parameters in relation to the opposing demands of high image quality and reasonably low patient radiation dose.
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    Open Access
    Study of the molecular mechanisms in piRNA-mediated gene regulation
    (2023-03-03) Κωνσταντινίδου, Παρθένα; Konstantinidou, Parthena
    From prokaryotic CRISPR to eukaryotic RNA interference (RNAi), small RNA-based mechanisms have been universally employed to defend the host against genome invaders, including viruses and transposable elements. Transposons and endogenous retroviruses are genomic DNA sequences that can change their position within the genome (transposition), causing mutagenesis and threatening genome stability. In animal gonads, a germline-specific class of small RNAs, associate with the PIWI subfamily of Argonaute proteins (PIWI-interacting RNAs, piRNAs) and guide the suppression of transposable elements on both transcriptional and post-transcriptional level. From invertebrates to mammals, the piRNA pathway is essential for germ cell development and fertility. During piRNA biogenesis, large genomic loci (piRNA clusters) produce long single stranded RNA precursors which are processed into thousands of unique piRNA sequences in a non-predictable manner. As a result, millions of different piRNAs have been observed in flies and mice. This remarkable diversity combined with the tolerated mismatches during target-recognition, pose a challenge to understand the non-self specificity of the piRNA-mediated genome defense. Here we investigated mechanisms of piRNA-guided transcriptional silencing through a combination of molecular, genetic, and computational approaches. We particularly aimed to understand how the diverse population of piRNAs avoids off-target events that would permanently shut down essential host genes. To accomplish this goal, we developed a quantitative piRNA-sequencing method and a direct reporter assay to measure piRNA-guided silencing at single cell level. Our work revealed that universally the abundance of unique piRNA sequences is highly skewed and only a few of the most-abundant piRNAs are present in every cell. We observe that individual piRNA abundance is regulated during the biogenesis step and is determined by the sequence preferences of the Zucchini processor. Our direct reporter assay demonstrated that efficient silencing of a target correlates with the combined abundance of antisense piRNAs and allowed us to observe cell to cell differences in piRNA-mediated transcriptional silencing. The vast majority of the diverse piRNA sequences occur sporadically and establish cell to cell diversity. Biogenesis of these sporadic piRNAs could be indicative of a mechanism that allows adaptation to new invaders. Furthermore, by establishing cell to cell diversity, piRNAs could be promoting reproductive polymorphism and increase the chances of species survival under evolutionary pressure. A direct consequence of our study is that piRNA abundance becomes a key consideration when studying mechanisms of piRNA function, biogenesis and targeting. Our results also emphasized the importance of high-quality, quantitative piRNA datasets that accurately distinguish between rare and abundant piRNA sequences. Thus, during this PhD, we also established a simple and reliable method for the preparation of high-quality piRNA sequencing libraries. Our method has eliminated the need for radioactive labeling and UREA-polyacrylamide gel purification, and provided an unbiased view of the complete bona fide piRNA population in Drosophila. With our improved method, we characterized Piwi-piRNA complexes in the different cell lineages of the Drosophila ovary. We observe that the same PIWI protein targets different transposons in the germ cells and in the follicle cells of the ovary. Upon association with a piRNA, this PIWI protein enters the nucleus and is responsible for the de novo epigenetic silencing of active transposons. Follicle PIWI-piRNA complexes target almost exclusively a single family of retrotransposons, namely Gypsy, while the corresponding germ cell complexes target a broader range of retrotransposon families and different members of the Gypsy family. This observation is linked to the differential expression of piRNA-generating clusters in the two ovarian cell types. How this differential expression of piRNA-generating regions is initially established and how are the transposons that are neglected by the piRNA pathway in the different compartments controlled, are open questions that would require further investigation. In summary my PhD work provides novel insight into key questions regarding the piRNA-mediated transcriptional silencing and it created resources and foundations for future piRNA studies.