Presente y futuro de la anología

ResumenEl advenimiento de la fecundación in vitro cambió radicalmente el pronóstico del factor masculino en relación a su participación en la esterilidad conyugal, mejorando sustancialmente las tasas de embarazo por ciclo con la aplicación de la técnica de ICSI. No obstante, otras situaciones serán las que tendrán que evaluarse en el futuro, como es el incremento del número de pacientes que desean preservar su fertilidad, ya sea por motivos oncológicos, de fertilidad, por portar enfermedades infecciosas trasmitidas por vía sexual, especialmente virus (inmunosupresores (HIV-SIDA), inductores de daño inflamatorio (Hepatitis B) ylo oncológicos (HPV); o que se someten en forma voluntaria a la esterilización quirúrgica, debiéndose contar para ello con métodos de criopreservación. En el área de la criobiología, la utilización de técnicas de congelación ultrarrápida ha permitido en forma exitosa preservar ovocitos, embriones y tejido ovárico; y en forma reciente este método se está utilizando para preservar el gameto masculino.SummaryThe advent of in vitro fertilization radically changed the prognosis of the male factor in relation to their participation in conjugal sterility, this substantially improves pregnancy rates per cycle with the application of the technique of ICSI. However, other situations will be evaluated in the future, such as the increase in the number of patients who wish to preserve their fertility, whether for cancer, fertility, because of being a carrier of infectious diseases transmitted through sex, especially viruses (immunosuppressant (HIV-AIDS), inducing inflammatory damage (Hepatitis B) and/ or cancer (HPV) or voluntarily submit to surgical sterilization, doing this with methods of cryopreservation in the area of cryobiology. In the area of cryobiology, the use of ultra rapid freezing techniques has allowed successfully preserve oocytes, embryos and ovarian tissue and in recent form this method is being used to preserve the male gamete

The p7 Protein of Hepatitis C Virus Forms Structurally Plastic, Minimalist Ion Channels

Hepatitis C virus (HCV) p7 is a membrane-associated oligomeric protein harboring ion channel activity. It is essential for effective assembly and release of infectious HCV particles and an attractive target for antiviral intervention. Yet, the selfassembly and molecular mechanism of p7 ion channelling are currently only partially understood. Using molecular dynamics simulations (aggregate time 1.2 ms), we show that p7 can form stable oligomers of four to seven subunits, with a bias towards six or seven subunits, and suggest that p7 self-assembles in a sequential manner, with tetrameric and pentameric complexes forming as intermediate states leading to the final hexameric or heptameric assembly. We describe a model of a hexameric p7 complex, which forms a transiently-open channel capable of conducting ions in simulation. We investigate the ability of the hexameric model to flexibly rearrange to adapt to the local lipid environment, and demonstrate how this model can be reconciled with low-resolution electron microscopy data. In the light of these results, a view of p7 oligomerization is proposed, wherein hexameric and heptameric complexes may coexist, forming minimalist, yet robust functional ion channels. In the absence of a high-resolution p7 structure, the models presented in this paper can prov

Palmitoylation of a processed form of hepatitis C virus core protein by host dhhc enzymes

Il a récemment été démontré que la nucléocapside du virus de l’hépatite C (VHC) subit l’ajout post-traductionnel d’un groupement acyl gras sur le résidu C172. Cette modification, appelée palmitoylation, est requise pour la production de virions infectieux. Chez les eucaryotes, la palmitoylation est catalysée par les enzymes de la famille DHHC. Notre objectif était d’identifier lesquelles des 23 protéines DHHC présentes chez les mammifère possèdent une activité palmitoyl-acyl transferase (PAT) sur la nucléocapside du VHC. Nous avons d’abord étudié l’expression et la localisation des protéines DHHC dans les hépatocytes humaines. Nous avons ensuite mesuré la variation du niveau de palmitoylation de la nucléocapside lorsque chaque DHHC candidate est surexprimée ou réprimée. Ce criblage a mené à l’identification de 5 enzymes qui exercent une activité PAT sur la nucléocapside du VHC : DHHC 1, 2, 3, 6 et 7. Leur co-localisation avec le substrat viral a été confirmée.As previously demonstrated, a fatty acid group is post-translationnally added on the residue C172 of Hepatitis C virus (HCV) core protein and this modification, termed palmitoylation, is required for viral assembly. In eukaryotes, palmitoylation is performed by a family of enzymes sharing a closely related DHHC motif. The purpose of this study was to identify which of the 23 mammalian DHHCs could perform palmitoyl acyltransferase activity on the HCV core protein. First, we characterized the expression and localization of DHHC enzymes in human hepatocytes. Then we evaluated the variation in core palmitoylation levels when each cellular DHHC proteins was over-expressed or repressed. This screen identified five enzymes with PAT activity on HCV core protein; DHHC 1, 2, 3, 6 and 7. Their co-localization with the viral protein was also demonstrated. These findings pave the way for future studies on the role of core palmitoylation during the HCV infection

Μελέτη της πολυμορφίας του HCV στην Ελλάδα: αναζήτηση σπανίων στελεχών και μέθοδοι προσδιορισμού τους

Η ανακάλυψη και ταυτοποίηση του ιού HCV ως τον αιτιολογικό παράγοντα των περιστατικών μη-Α, μη-Β ηπατίτιδας ήταν η αρχή για την ανάπτυξη διαφόρων μεθόδων και τεχνολογιών για την ανίχνευση και τυποποίησή του. Ο ιός HCV ανήκει στην οικογένεια Flaviviridae, γένος hepacivirus και το γονιδίωμά του είναι μονόκλωνο RNA θετικής πολικότητας που κωδικοποιεί μια πολυπρωτεϊνη από την οποία προκύπτουν οι δομικές και οι μη δομικές πρωτεϊνες του. Κύτταρα-στόχοι του ιού – στα οποία εισέρχεται με ενδοκύτωση μετά από πρόσδεση σε ειδικούς υποδοχείς- είναι τα ηπατοκύτταρα αλλά αναφέρονται και τα λεμφοκύτταρα (Β και Τ) καθώς και τα δενδριτικά και τα μονοκύτταρα. Παρόλο που το ανοσοποιητικό σύστημα καταφέρνει να περιορίσει τη λοίμωξη και να οδηγήσει σε αυτοϊαση, εντούτοις ένα μεγάλο ποσοστό ασθενών (80-85%) μεταπίπτουν σε χρονιότητα από τους οποίους το 5-25% θα φθάσουν σε στάδιο κίρρωσης διατρέχοντας τον κίνδυνο εμφάνισης ηπατικής νόσου τελικού σταδίου και ηπατοκυτταρικό καρκίνο. Ο ιός HCV παρουσιάζει μεγάλη γενετική ετερογένεια, έχει παγκόσμια εξάπλωση και έχει μολύνει πάνω από 150 εκ. ανθρώπους στην υφήλιο. Διακρίνεται σε 7 γονότυπους και πολλούς υπότυπους. Η ανίχνευση και ο ποσοτικός προσδιορισμός του HCV RNA δίνουν πολύτιμες πληροφορίες τόσο για τη διάγνωση της χρόνιας λοίμωξης όσο και για την παρακολούθηση της πορείας της θεραπείας. Επιπλέον, η γνώση του γονότυπου του ιού με τον οποίο είναι μολυσμένος ο ασθενής καθορίζει τη δόση και τη διάρκεια του θεραπευτικού σχήματος και αποτελεί πολύτιμο εργαλείο για την παρακολούθηση και τη δυναμική της επιδημίας της ηπατίτιδας C σε διάφορους πληθυσμούς. Σκοπός της παρούσας διατριβής ήταν η ανάπτυξη in-house μεθόδων α) για την ανίχνευση και τον ποσοτικό προσδιορισμό του HCV RNA και β) για τον προσδιορισμό του γονότυπου του ιού HCV προκειμένου να μελετηθεί η πολυμορφία του HCV στην Ελλάδα. Σε ότι αφορά στη μέθοδο του ποσοτικού προσδιορισμού του HCV RNA στοχεύσαμε το 3΄άκρο του γονιδιώματος του ιού σε αντίθεση με όλες τις εμπορικά διαθέσιμες μεθόδους που βασίζονται στην ενίσχυση του 5΄άκρου. Μετά από πολλές δοκιμές με διάφορες πλατφόρμες και συνδυασμούς αντιδραστηρίων τελικά καταλήξαμε στη χρησιμοποίηση της τεχνολογίας της one step real time PCR (αλυσιδωτή αντίδραση της πολυμεράσης πραγματικού χρόνου σε ένα βήμα) στην πλατφόρμα Light Cycler (Roche) και η αξιοπιστία της μεθόδου ελέγχθηκε τόσο με διεθνή standards όσο και με τη σύγκρισή της με δύο εγκεκριμένες εμπορικές μεθόδους των εταιρειών Abbott και Roche. Έτσι δημιουργήθηκε μια μέθοδος που είναι γρήγορη, αξιόπιστη και χαμηλού κόστους και η οποία μπορεί να βελτιωθεί περαιτέρω. Σε ότι αφορά στη μέθοδο γονοτύπησης η μέθοδος που αναπτύχθηκε ήταν ο άμεσος προσδιορισμός της αλληλουχίας με τη διαδικασία του sequencing που αποτελεί και τη μέθοδο αναφοράς. Αρχικά σχεδιάστηκαν εκκινητές (primers) από την πολύ καλά συντηρημένη περιοχή NS5B του ιού και δοκιμάστηκαν διάφορα πρωτόκολλα PCR (αλυσιδωτής αντίδρασης της πολυμεράσης). Ακολούθησε η διαδικασία του sequencing για τη λήψη των αλληλουχιών οι οποίες στη συνέχεια επεξεργάζονταν με μεθόδους φυλογενετικής ανάλυσης. Η μέθοδος αξιολογήθηκε με διεθνή πρότυπα και έλαβε πιστοποίηση από το ΕΣΥΔ. Με τη μέθοδο αυτή τυποποιήθηκαν τα περισσότερα δείγματα που ήταν απροσδιόριστα (με τη μέθοδο του αντίστροφου υβριδισμού (LiPA)) στο εργαστήριό μας και ήταν η μέθοδος με την οποία μελετήθηκε η δυναμική της επιδημίας της ηπατίτιδας C σε ΧΕΝ (χρήστες ενδοφλεβίων ναρκωτικών) στα πλαίσια του προγράμματος «Αριστοτέλης» τα δεδομένα της οποίας δημοσιεύτηκαν σε έγκυρο επιστημονικό περιοδικό.The discovery of the HCV virus as the etiologic factor for the non A, non B hepatitis cases, led to the development of many methods and technologies for its detection and identification. HCV is a flavivirus and its genome is a single stranded RNA molecule which encodes for a polyprotein which provides the structural and non structural proteins of the virus. The target cells for HCV are the hepatocytes as well as B and T lymphocytes, dendritic and mononuclear cells which the virus enters throygh endocytosis. Although the immune system manages to clear the virus a large number (80-85%) of patients become chronic carriers from which about 5-25% will develop chirrossis, end stage liver disease and hepatocellular carcinoma. HCV has a great genetic diversity, is spread worldwide having infected more than 150 million of people and is classified in seven major genotypes and many subtypes. The detection and quantification of thw HCV RNA are valuable tools for monitoring the chronic infection and the outcome of the therapy. Furthermore, knowing the genotype with which a patient is infected is crucial for the choice of the therary regimen. The aim of this study was to develop two in house methods a) for the detection and quantification of the HCV RNA and b) for the determination of the genotype in order to study the diversity of HCV in Greece. For the quantification method we targeted at the 3’UTR end of the genome in contrast to all the commercially available methods which target the 5’end. After experimenting with different types of reagents and enzymes and on different platforms we finally chose the technology of one step real time RT PCR on the Light Cycler platform (Roche). The method was validated with international standards and was evaluated with two commercially available methods from Abbott and Roche with excellent results. The method is quick, reliable and low cost and can be further developed. For the HCV genotyping we developed a method based on the sequencing of the NS5B region of the genome. We designed primers and tried many PCR protocols. The obtained sequences were analysed with phylogenetic analysis methods. The method was validated with international standards and was certified by ΕΣΥΔ. With this method we genotyped most of the undetermined samples (by other methods) in our lab and mainly we studied the HCV dispersal pattern among IDUs (intravenous drug users) in Athens as part of the “Aristotle” programme

Exploration of new uracil-based compounds as novel inhibitors of Hepatitis C Virus replication

Hepatitis C Virus (HCV) is a major public health problem worldwide. While highly efficacious directly-acting antiviral agents have been developed in recent years, their high costs and relative inaccessibility make their use limited. In this thesis, new uracil-based compounds have been evaluated as potential antiviral drugs against HCV. Using several biochemical and virological assays to investigate virus infection and replication, it has been shown that these compounds are able to significantly reduce viral genomic replication with their IC50 values in the nanomolar range. Finally, these compounds have been shown to block the de novo RNA synthesis and that effect is dependent on a chemical structure of the compounds

The SAT Protein of the Minute Virus of Mice Induces the Lysis of the Cell through the Formation of Viroporin-like Structures

The prototype minute virus of mice (MVMp) and the H-1 virus belong to the family of Parvoviridae and can be used as oncolytic agents that infect, replicate in and kill human cancer cells. In preclinical trials the viruses were shown to be very successful to eradicate tumors in mice and rats. A phase I/IIa clinical trial with patients suffering from recurrent glioblastoma multiforme showed that parvovirus is safe to use in humans and that the therapy induces an infiltration of immune cells into the tumor tissue. Many aspects of the parvoviral life cycle have been studied over the last decades. The entry, replication, transcription and packaging mechanisms of the virus are well understood. However, the process of the virally-induced lysis of the cell is still unknown. The viral non structural protein 1 (NS1) was suggested to have cytotoxic features but a direct lytic mechanism could not be shown. Recently, the porcine parvovirus (PPV) was found to express the short transmembrane protein SAT. Mészáros and coworkers showed that a knockout of SAT led to a reduced lytic capacity of the virus (Mészáros et al., 2017). In the current study we show that the SAT protein of MVMp is also important for the lysis of the cell. A knockout of SAT in the genome of MVMp reduced the lytic capacity of the virus and SAT-ko viruses were also found to be less infectious than the wild type virus. The sole expression of SAT induced lysis and it exceeded the lytic capacity of NS1 by multiple times. Mészáros and coworkers suspected SAT to induce an irreversible stress in the endoplasmic reticulum which eventually led to the death of the cell. However, a direct mechanism of this process could not be shown. We suspect SAT to function as a viroporin - a class of virally-encoded transmembrane proteins that oligomerise to form pores through membranes. We could show that SAT is transported to the plasma membrane where it oligomerises in multimers and makes the plasma membrane permeable to the small molecule Hygromycin B. A computer simulation of the protein confirmed that SAT oligomerises in symmetrical complexes. However, pore-like structures were not observed. Furthermore, an increase in the permeability of ions such as calcium and sodium, which is often seen for other viroporins, was not found. In an attempt to increase the lytic capacity of MVMp we created the SUPER virus. The position of SAT was altered in the genome of SUPER in order to increase its translation. We could show that the translation of SAT was indeed increased for the SUPER virus which led to an accelerated lysis of the cells. However, the production of the SUPER virus was not very efficient. In order to increase the production of SUPER, we constructed shRNA constructs to knockdown SAT. Although the production of SAT was decreased, this approach did not increase the production of the virus. Nevertheless, the SUPER virus could be a promising candidate for the therapy with oncolytic parvoviruses. We suspect its increased lytic potential to release tumor antigens more efficiently compared to the lytic potential of the wild type virus. The released tumor antigens are suspected to stimulate cells of the immune system such as dendritic cells and cytotoxic T-cells in order to recognise and attack non-infected tumor cells

Analysis of the role of p7 protein function in hepatitis C virus life cycle

The hepatitis C virus genome encodes a 63 amino acid protein, p7. The exact role of p7 is unknown; in this study we observed that mutations in transmembrane domain-1 and -2 (TM1 and TM2), and the cytoplasmic loop of p7 decreased infectious virus production. Analysis of p7 at different stages of virus assembly revealed that p7 functions at a stage prior to generation of infectious particles. Confocal microscopy analyses indicated that p7 did not affect recruitment of core protein to lipid droplets. Additionally, mutation of p7 did not affect formation of core multi-order structures. Finally, mutations at the cytoplasmic loop significantly reduced intracellular E2 glycoprotein levels. Forced evolution analysis of p7 mutations resulted in the occurrence of a N765D mutation that was important for secretion of virus particles into the culture supernatants. The results of this study provide strong evidence that p7 functions to protect HCV glycoproteins from premature degradation and we suggest that p7 supports the virus assembly process