Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. Previously, it has been shown that the RNA released from damaged blood cells activates clotting. However, the nature of RNA released from hemolysis is still elusive. We found that after hemolysis, the red blood cells from both zebrafish and humans release 5.8S rRNA. This RNA activated coagulation in zebrafish and human plasmas. Using both natural and synthetic 5.8S rRNA and its synthetic truncated fragments, we found that the 3'-end 26 nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor blocked 3'-26 RNA-mediated coagulation activation of both zebrafish and human plasma. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activates normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via FXII-like protein. Since zebrafish has no FXII and hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from this knockdown fish does not respond to 3'-26 RNA. In conclusion, we identified 5.8S rRNA released in hemolysis activates clotting in human and zebrafish plasma. Only 3'-end 26 nucleotides of the 5.8S rRNA is needed for the clotting activity. Furthermore, we showed that fish Hgfac plays a role in 5.8S rRNA-mediated activation of coagulation.

Abstract We designed a 16S rRNA gene PCR-RFLP scheme to identify all currently described Bartonella spp. The 16S rRNA genes of all Bartonella spp. were in-silico analyzed in order to design a RFLP technique able to discriminate among different species. The restriction enzymes selected were MaeIII, MseI, Sau96I, BsaAI, DrdI, FokI, BssHII, BstUI, AluI, TspDTI and HphI which, according to a decision-making tree, facilitated the differentiation of all the currently described species of Bartonella.The technique was experimentally tested in different species of Bartonella, including human pathogenic B. bacilliformis and B. henselae with a 100% of concordance with the in-silico predicted patterns.This novel RFLP assay could be used to identify both human and non-human pathogenic Bartonella in diagnostic, phylogenetic and epidemiologic studies.

From its introduction in 1952 onwards, the clinical use of macrolides has been steadily increasing, both in human and veterinary medicine. Although initially designed to the treatment of Grampositive microorganisms, this antimicrobial family has also been used to treat specific Gram-negative bacteria. Some of them, as azithromycin, are considered in the armamentarium against Enterobacteriaceae infections. However, the facility that this bacterial genus has to gain or develop mechanisms of antibiotic resistance may compromise the future usefulness of these antibiotics to fight against Enterobacteriaceae infections. The present review is focused on the mechanisms of macrolide resistance, currently described in Enterobacteriaceae.

Parodontitis ist eine Erkrankung des Zahnhalteapparates, die durch einen komplexen bakteriellen Biofilm unterhalten wird. Neben Mikroorganismen wie A. actinomycetemcomitans, P. gingivalis, T. denticola und T. forsythensis werden Keime unbekannter Spezies in parodontalen Taschen ausfindig gemacht. Durch die Entschlüsselung von 16S rRNA-Gensequenzen konnte die orale Flora nahezu vollständig katalogisiert werden. Allerdings fehlen bei vielen Phylotypen die entsprechenden Typstämme für weitergehende phänotypische Analysen. Grundlage dieser Arbeit bildeten 59 Patientenisolate der Parodontitis-Stammsammlung des Instituts für Hygiene und Mikrobiologie bei denen partielle 16S rRNA Sequenzen keine Spezieszuordnung ermöglichten. Nahezu vollständige 16S rRNA-Sequenzen wurden erstellt und mit Datenbankeinträgen verglichen. Bei mehr als der Hälfte der Stämme konnte keine taxonomische Zuordnung auf Sequenzierebene getroffen werden. 43 Isolate wuchsen unter aerober Atmosphäre, 16 benötigten eine anaerobe Umgebung. Alle Kulturmorphologien und nach Gram gefärbten mikroskopischen Präparate wurden fotografisch dokumentiert und katalogisiert. Die hier untersuchten Stämme, die zufällig auf der Basis taxonomischer Fragestellungen ausgewählt wurden, waren zum überwiegenden Teil auf Amoxicillin und Metronidazol empfindlich. Diese Antibiotika finden alle ihre Verwendung bei der Parodontitistherapie. Ciprofloxacin, das wegen seiner intrazellulären Wirkung ein interessantes Agens ist, wies v.a. bei Actinomyceten und Streptokokken Wirkungslücken auf. Es bleibt zu diskutieren, ob dieser Umstand nachteilig ist, da auf der einen Seite diese Genera ein orales Reservoir für Gyrasehemmer-Resistenzen ausbilden können, auf der anderen Seite diese grampositiven Keime möglicherweise parodontalprotektiv wirken könnten. In dieser Studie konnte eine Stammsammlung charakterisiert werden, die zukünftig insbesondere angesichts der zu erwartenden Entschlüsselung des oralen Metagenoms für weitere funktionelle Untersuchungen von Interesse sein dürfte. / The aim of this study was the characterisation of the subgingival periodontal microbiota by cultural and molecular methods. Periodontitis is a bacterial disease, which is caused by different periodontal pathogens like Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythensis. Furthermore there exist some species in the complex subgingival biofilm that are not cultured yet. They may participate in the progression or persistence of periodontal destruction. In this paper 59 Plaque samples were obtained from a previous study of the Institute for Hygiene and Microbiology from the University of Würzburg where the comparison of subgingival microorganisms with public databases didn´t allow a species identification. Almost complete 16S rRNA sequences were provided and compared with database entries. More than half of the bacterial isolates didn’t permit a taxonomic allocation. Macroscopic and microscopic features, the susceptibility against amoxicillin, ciprofloxacin and metronidazole are described here. 43 isolates grew under aerobic conditions, 16 needed anaerobic atmosphere. The morphology of the culture and the colouring according to Gram were documented photographically and listed. Most tested microorganisms were susceptible to amoxicillin and metronidazole. Except for Actinomyces and Streptococcus ciprofloxacin exhibits good results in the treatment of periodontal disease. In this study a collection of subgingival bacteria could be characterized, which might be of interest for further investigations in view of the decoding of the oral metagenome.

A síntese de ribossomos é um dos principais processos celulares e na levedura Saccharomyces cerevisiae são necessários 75 snoRNAs e mais de 200 proteínas não-ribossomais para que o ribossomo seja corretamente formado. Para o processamento do precursor dos RNAs ribossomais, chamado pré-rRNA 35S, ocorre o pareamento deste com o U3 snoRNA e outros snoRNAs e diversas proteínas se associam de maneira orquestrada e transitória, formando o complexo SSU processomo. Tal complexo é necessário para o processamento da região 5\' do pré-rRNA 35S e para a correta montagem e maturação da subunidade menor ribossomal. Estudos anteriores do nosso laboratório identificaram a proteína nucleolar Utp25, essencial em S. cerevisiae, como integrante do complexo SSU processomo. Foi demonstrado que a depleção de Utp25 afeta a formação da subunidade menor ribossomal e que Utp25 interage com as proteínas Sas10 e Mpp10, componentes do SSU processomo, além de Utp25 co-imunoprecipitar o snoRNA U3. A partir desses dados, este trabalho teve como objetivo identificar interações da proteína Utp25 com outros componentes do complexo SSU processomo e investigar o papel de tais interações na formação e funcionamento do mesmo. Para purificação do complexo SSU processomo nós utilizamos o método Tandem Affinity Purification-tag (TAP-tag) utilizando TAP-Utp25 como isca. Após análise do purificado resultante por espectrometria de massas, obtivemos como resultado as proteínas Rrp5, Snu13 e Nop56, sendo as duas últimas pertencentes ao subcomplexo U3 snoRNP. / The ribosome synthesis is one of the main cellular processes and in the yeast Saccharomyces cerevisiae 75 snoRNAs and more than 200 non-ribosomal proteins are involved in ribosome maturation. During processing, the pre-rRNA 35S base pairs with the U3 snoRNA and other snoRNAs and several proteins associate, forming the SSU processome complex. This complex is required for the processing of the pre-rRNA 35S 5\' region and for the correct assembly and maturation of the ribosome small subunit. Previous studies from our laboratory identified the nucleolar protein Utp25, essential in S. cerevisiae, as a member of the SSU processome complex. Utp25 depletion affects small ribosomal subunit formation. Utp25 interacts with proteins Sas10 and Mpp10, components of the SSU processome, and Utp25 co-immunoprecipitates U3 snoRNA. From these data, this study aimed to identify Utp25 interactions with other components of the SSU processome complex and to investigate the role of these interactions in this complex formation and function. For the SSU processome complex purification we used the Tandem Affinity Purification-tag method (TAP-tag) and TAP-Utp25 as the bait. After the resulting purified analysis by mass spectrometry, we obtained as results the Rrp5, Snu13 and Nop56 proteins, the last two being U3 snoRNP subcomplex components.

Optimizing the production of useful macromolecules from transgenic microorganisms is crucial to biopharmaceutical companies. Improving bacterial growth and replication depends largely on the efficiency of translation, which is rate-limited by initiation. Among the most important interactions between the mRNA translation initiation region (TIR) and the translation machinery is the association between the Shine-Dalgarno (SD) sequence in the TIR and the complementary anti-SD (aSD) sequence which is located within a short unstructured segment that includes the 3’ terminus (3’ TAIL) of the mature 16S rRNA. However, the mature 3’ TAIL has been poorly characterized in the majority of bacteria, rendering optimal SD/aSD pairing unclear in these species. In light of this, we established a novel strategy to characterize the mature 3’ TAILs of bacterial species that leverages the availability of publically stored RNA sequencing (RNA-Seq) data. In chapter 2, we devised a RNA-Seq-based approach to successfully recover the experimentally verified 3’ TAIL in E. coli (5’-CCUCCUUA-3’) and resolve inconsistencies surrounding the identity of the 3’ TAIL in Bacillus subtilis. In chapter 3 we improve the method introduced in chapter 2 to clearly and more reliably define the 3’ TAIL termini for 13 bacterial species with available protein abundance data. Our results reveal considerable heterogeneity in the termini of 3’ TAILs among closely related species and that sites downstream of the canonical CCUCC aSD motif are more important to initiation than previously believed. My research contributes to advance our understanding in microbial translation efficiency in two significant ways: 1) providing an RNA-Seq-based approach to characterize rRNA transcripts, and 2) elucidating optimal recognition between protein-coding genes and the rRNA translation machinery.

A síntese de ribossomos é um dos principais processos celulares e na levedura Saccharomyces cerevisiae são necessários 75 snoRNAs e mais de 200 proteínas não-ribossomais para que o ribossomo seja corretamente formado. Para o processamento do precursor dos RNAs ribossomais, chamado pré-rRNA 35S, ocorre o pareamento deste com o U3 snoRNA e outros snoRNAs e diversas proteínas se associam de maneira orquestrada e transitória, formando o complexo SSU processomo. Tal complexo é necessário para o processamento da região 5\' do pré-rRNA 35S e para a correta montagem e maturação da subunidade menor ribossomal. Estudos anteriores do nosso laboratório identificaram a proteína nucleolar Utp25, essencial em S. cerevisiae, como integrante do complexo SSU processomo. Foi demonstrado que a depleção de Utp25 afeta a formação da subunidade menor ribossomal e que Utp25 interage com as proteínas Sas10 e Mpp10, componentes do SSU processomo, além de Utp25 co-imunoprecipitar o snoRNA U3. A partir desses dados, este trabalho teve como objetivo identificar interações da proteína Utp25 com outros componentes do complexo SSU processomo e investigar o papel de tais interações na formação e funcionamento do mesmo. Para purificação do complexo SSU processomo nós utilizamos o método Tandem Affinity Purification-tag (TAP-tag) utilizando TAP-Utp25 como isca. Após análise do purificado resultante por espectrometria de massas, obtivemos como resultado as proteínas Rrp5, Snu13 e Nop56, sendo as duas últimas pertencentes ao subcomplexo U3 snoRNP. / The ribosome synthesis is one of the main cellular processes and in the yeast Saccharomyces cerevisiae 75 snoRNAs and more than 200 non-ribosomal proteins are involved in ribosome maturation. During processing, the pre-rRNA 35S base pairs with the U3 snoRNA and other snoRNAs and several proteins associate, forming the SSU processome complex. This complex is required for the processing of the pre-rRNA 35S 5\' region and for the correct assembly and maturation of the ribosome small subunit. Previous studies from our laboratory identified the nucleolar protein Utp25, essential in S. cerevisiae, as a member of the SSU processome complex. Utp25 depletion affects small ribosomal subunit formation. Utp25 interacts with proteins Sas10 and Mpp10, components of the SSU processome, and Utp25 co-immunoprecipitates U3 snoRNA. From these data, this study aimed to identify Utp25 interactions with other components of the SSU processome complex and to investigate the role of these interactions in this complex formation and function. For the SSU processome complex purification we used the Tandem Affinity Purification-tag method (TAP-tag) and TAP-Utp25 as the bait. After the resulting purified analysis by mass spectrometry, we obtained as results the Rrp5, Snu13 and Nop56 proteins, the last two being U3 snoRNP subcomplex components.

Τα τελευταία χρόνια, πολλά εργαστήρια έχουν επικεντρώσει το ενδιαφέρον τους στο 5S rRNA, το μικρότερο RNA συστατικό του ριβοσώματος. Ο ακριβής ρόλος του 5S rRNA στο ριβόσωμα, την πρωτεϊνική βιοσυνθετική μηχανή, δεν έχει πλήρως αποσαφηνιστεί. Αποτελέσματα δομικών μελετών τοποθετούν το 5S rRNA στη μεγάλη ριβοσωμική υπομονάδα. Ευρήματα που αφορούν σχέσεις δομής και λειτουργίας οδηγούν στην υπόθεση, ότι σταθεροποιεί τη δομή του ριβοσώματος και λειτουργεί ως φυσικός μεταδότης πληροφοριών μεταξύ λειτουργικών κέντρων. Από την άλλη πλευρά, το ιοντικό περιβάλλον επηρεάζει άμεσα τη ριβοσωματική λειτουργία. Μονοσθενή και δισθενή κατιόντα, καθώς και πολυκατιόντα, όπως οι πολυαμίνες, παρεμβαίνουν στη ριβοσωματική διαμόρφωση και συνεπώς εμπλέκονται άμεσα στη λειτουργία του ριβοσώματος. Προκαρτατικές μελέτες στο εργαστήριό μας έχουν δείξει ότι οι πολυαμίνες, και συγκεκριμένα η σπερμίνη, σταθεροποιούν την δευτεροταγή και τριτοταγή δομή του 5S rRNA, οδηγώντας σε μια πιο συνεκτική διαμόρφωση του μορίου. Στόχος της παρούσας διατριβής ήταν να διερευνηθεί ο ρόλος της διαμόρφωσης του 5S rRNA στην πρωτεϊνική σύνθεση του εντεροβακτηρίου E.coli. Παράλληλος στόχος ήταν η μελέτη της επίδρασης των πολυαμινών στη δομή και λειτουργία τόσο του 5S rRNA, όσο και ολοκλήρου του ριβοσωματικού συμπλόκου. Η πρόσδεση των πολυαμινών στο 5S rRNA μελετήθηκε με τη μέθοδο της φωτοσήμανσης συγγένειας, χρησιμοποιώντας ως μοριακό ανιχνευτή ένα φωτοδραστικό ανάλογο της σπερμίνης, την ΑΒΑ-σπερμίνη. Πραγματοποιήθηκε κινητική μελέτη της πρόσδεσης και προσδιορίστηκαν οι θέσεις δέσμευσης της ΑΒΑ-σπερμίνης στο 5S rRNA, είτε αυτό ήταν ελεύθερο, είτε ενσωματωμένο σε 50S ριβοσωματικές υπομονάδες ή 70S ριβοσωματικά σύμπλοκα από E.coli. Τα αποτελέσματα αποκάλυψαν ότι σε ελεύθερο 5S rRNA, η σπερμίνη προσδένεται κυρίως σε νουκλεοτίδια των ελίκων III και V και στις θηλιές Α, C, D και E. Ενσωμάτωση του 5S rRNA σε 50S υπομονάδες ή 70S poly(U)-προγραμματισμένα ριβοσώματα, είχε σαν αποτέλεσμα τη μείωση της επιδεκτικότητας των νουκλεοτιδίων του στην ΑΒΑ-σπερμίνη. Ακολούθησε διερεύνηση των επιπτώσεων της πρόσδεσης του φωτοαναλόγου στη δομή του 5S rRNA. Πραγματοποιήθηκαν πειράματα χημικής τροποποίησης, τα οποία αποκάλυψαν ότι ανεξάρτητα από το αν το 5S rRNA είναι ελεύθερο ή συναρμολογημένο σε 50S υπομονάδες ή σε 70S ριβοσωματικά σύμπλοκα, η πρόσδεση της ΑΒΑ-σπερμίνης προκαλεί σημαντικές αλλαγές στη διαμόρφωση του μορίου. Η θηλιά Α υιοθετεί μια πιο χαλαρή δομή, ενώ οι θηλιές C, D και E και η έλικα III μια πιο συνεκτική δομή. Με την διαπίστωση των αλλαγών της διαμόρφωσης του 5S rRNA λόγω της πρόσδεσης της ΑΒΑ-σπερμίνης, κρίθηκε ενδιαφέρον να μελετηθεί αν αυτές οι διαμορφωτικές αλλαγές επηρεάζουν θεμελιώδεις λειτουργίες του ριβοσώματος. Αρχικά μελετήθηκε η επίδραση της αλλαγής διαμόρφωσης του 5S rRNA στην ικανότητα του να συναρμολογείται σε 50S υπομονάδες και 70S ριβοσωματικά σύμπλοκα. Τα αποτελέσματα έδειξαν ότι η πρόσδεση της ΑΒΑ-σπερμίνη στο 5S rRNA, δεν καταργεί την διαδικασία συναρμολόγησης των 50S υπομονάδων, παρεμποδίζει όμως τις απαιτούμενες δομικές αλλαγές με το να σταθεροποιεί μια πιο συνεκτική δομή του 5S rRNA, θερμοδυναμικά λιγότερο ευνοϊκή για την πορεία. Παρά το γεγονός ότι η πρόσδεση της σπερμίνης στο 5S rRNA επιφέρει στο μόριο δομικές αλλαγές που εμποδίζουν την συναρμολόγηση του σε 50S υπομονάδες, όταν αυτές σχηματιστούν, αποκτούν δομή τέτοια που επιτρέπει την αλληλεπίδρασή τους με τις 30S υπομονάδες. Ωστόσο, η παρουσία του 5S rRNA φαίνεται να είναι απαραίτητη για τον σχηματισμό ενεργών 50S υπομονάδων, αφού η απουσία του 5S rRNA δεν επιτρέπει ούτε το σχηματισμό ακέραιων υπομονάδων, ούτε την σύζευξη των υπομονάδων προς ακέραια ριβοσωματικά σύμπλοκα. Στη συνέχεια μελετήθηκε η επίδραση της φωτοσήμανσης του 5S rRNA, αρχικά στην ικανότητα των ριβοσωμάτων να προσδένουν υπόστρωμα και στη συνέχεια, στην καταλυτική δράση αυτών. Φωτοσήμανση του 5S rRNA με ΑΒΑ-σπερμίνη προκάλεσε μικρή, αλλά σαφή ενεργοποίηση της πρόσδεσης υποστρώματος στην Ρ-θέση, σε αντίθεση με την πρόσδεση στην Α-θέση, η οποία δε φαίνεται να επηρεάζεται. Σαφής ήταν, επίσης, η επίδραση στην ενεργότητα της πεπτιδυλοτρανσφεράσης: Παρατηρήθηκε αύξηση μέχρι και 30% στη δραστικότητα της ΡΤασης. Ιδιαίτερης σημασίας είναι το εύρημα, ότι ριβοσώματα ελλιπή σε 5S διατηρούν ~10% της δράσης της ΡΤασης, γεγονός που οδηγεί στη υπόθεση ότι το 5S rRNA δεν είναι απολύτως απαραίτητο για τη κατάλυση του πεπτιδικού δεσμού. Ακολούθησε η μελέτη της επίδρασης της αλλαγής διαμόρφωσης του 5S rRNA στην μετατόπιση υποστρωμάτων, αρχικά στην αυθόρμητη (μη ενζυμική) και στη συνέχεια στην EF-G-καταλυόμενη μετατόπιση. Τα αποτελέσματα επιβεβαίωσαν την υπόθεση, ότι η μετατόπιση υποστρωμάτων είναι έμφυτη ιδιότητα του ριβοσώματος. Παράλληλα, κατέδειξαν ότι η πρόσδεση σπερμίνης στο 5S rRNA διεγείρει την μη ενζυμική μετατόπιση, πιθανόν μέσω αλλαγών στην αναδίπλωση του rRNA, οι οποίες μεταδίδονται στα ριβοσωματικά κέντρα που είναι υπεύθυνα για αυτή τη λειτουργία. Περαιτέρω, η παρουσία σπερμίνης στο 5S rRNA, μείωσε τις απαιτήσεις για EF-G. Το αποτέλεσμα αυτό μπορεί να εξηγηθεί είτε με μια πιθανή ευεργετική δράση της σπερμίνης στην δέσμευση του EF-G στο ριβόσωμα, είτε με την επίδρασή της στο στάδιο της υδρόλυσης του GTP. Για να διευκρινίσουμε ποια από τις δυο εκδοχές ευσταθεί, μελετήθηκε η επίδραση της αλλαγής διαμόρφωσης του 5S rRNA στην ικανότητα ριβοσωμάτων να δεσμεύουν EF-G και στη συνέχεια στην ικανότητα τους να ενεργοποιούν την EF-G-εξαρτώμενη GTP υδρόλυση. Βρέθηκε ότι, η παρουσία σπερμίνης στο μόριο του 5S rRNA, ευνοεί τη δέσμευση του EF-G στο ριβόσωμα. ¨Όσο αφορά την ικανότητα του ριβοσώματος να ενεργοποιεί την EF-G-εξαρτώμενη GTP υδρόλυση, τα αποτελέσματα της μελέτης έδειξαν ότι η αρχική ταχύτητα της αντίδρασης δεν επηρεάζεται από την παρουσία σπερμίνης, αποτέλεσμα που μας οδηγεί στο συμπέρασμα ότι η αλλαγή της διαμόρφωσης του 5S rRNA δεν επηρεάζει την GTP υδρόλυση. Απουσία του 5S rRNA, τα ριβοσώματα διατηρούν κάποια δραστικότητα GTPασης, αλλά δεν είναι ικανά να δεσμεύουν αποτελεσματικά τον EF-G, ακόμη και παρουσία σπερμίνης. Λαμβάνοντας υπόψη, ότι η θέση δέσμευσης του EF-G στο ριβόσωμα βρίσκεται στα domain II και VI του 23S rRNA και ότι απευθείας επαφές του 5S rRNA με αυτές τις περιοχές ή με τον EF-G δεν έχουν βρεθεί, οδηγούμαστε στην υπόθεση ότι, το 5S rRNA, μέσω αλλοστερικών σημάτων, επηρεάζει την EF-G-GTP δέσμευση στο ριβόσωμα, όχι όμως και την GTP υδρόλυση. Για να επιβεβαιώσουμε τα παραπάνω, μελετήθηκε η επίδραση του ιοντικού περιβάλλοντος και η αλλαγή της διαμόρφωσης του 5S rRNA, στη διαμορφωτική κατάσταση θεμελιωδών λειτουργικών κέντρων του ριβοσώματος. Η μελέτη εστιάστηκε στην ικανότητα του 5S rRNA να επηρεάζει τα κέντρα πρόσδεσης του EF-G στο ριβόσωμα, καθώς και τις θέσεις Α- και Ρ- του ριβοσωμικού συμπλόκου. Διαπιστώθηκε ότι η αλληλεπίδραση ελεύθερης σπερμίνης με το ριβόσωμα ενισχύει το αποτύπωμα (footprinting) χημικής προστασίας νουκλεοτιδίων υπευθύνων για την πρόσδεση του ΕF-G και τη συγκρότηση της Ρ-θέσης, όχι όμως και της Α-θέσης. Παρόμοια, αλλά μικρότερου βαθμού, ήταν και η επίδραση της προσδενομένης επιλεκτικά ΑΒΑ-σπερμίνης στο 5S rRNA. Συμπερασματικά, το 5S rRNA περιέχει θέσεις εξειδικευμένης πρόσδεσης πολυαμινών, οι οποίες επηρεάζουν την τριτοταγή του διαμόρφωση. Η δέσμευση της σπερμίνης στο 5S rRNA φαίνεται να μην επηρεάζει σημαντικά την ικανότητα της 50S ριβοσωματικής υπομονάδας να δεσμεύει υπόστρωμα, βελτιώνει όμως την καταλυτική δραστικότητα του ριβοσώματος και την ικανότητα του να μετατοπίζει τα υποστρώματα. Οι πολυαμίνες ενεργοποιούν την δέσμευση του EF-G στο ριβόσωμα, αλλά ελάχιστα επηρεάζουν την υδρόλυση του GTP από τον EF-G. Εν κατακλείδι, το 5S rRNA φαίνεται να παίζει σημαντικό ρόλο στην μεταγωγή σημάτων μεταξύ του καταλυτικού κέντρου και της περιοχής πρόσδεσης EFG. / In recent years, many research groups have focused their interest on 5S rRNA, the smallest RNA component of the ribosome that is preserved in almost all organisms. Its precise role in the ribosome, the protein “biosynthetic machine”, has not been fully clarified. The results of structural studies place 5S rRNA in the large ribosomal subunit. Findings concerning relations between structure and function lead to the assumption that 5S rRNA stabilises the structure of the ribosome and acts as natural transmitter of information between functional centres. On the other hand, the ionic environment directly affects ribosome function. Monovalent and divalent ions as well as polycations, such as polyamines, influence the ribosomal conformation, and therefore are involved in the function of the ribosome. Preliminary studies in our laboratory have shown that polyamines, namely spermine, stabilize the secondary and tertiary structure of 5S rRNA, leading to a more cohesive configuration of the molecule. The aim of this thesis was to investigate the role of 5S rRNA configuration in protein synthesis of the enterobacterium E.coli. At the same time, the biological activity of polyamines in the structure and functioning of both 5S rRNA and the whole ribosomal complex was studied. Polyamine binding to 5S rRNA was investigated by photoaffinity labeling, using a photoreactive analogue of spermine, ABA-spermine, as a molecular probe. Kinetic study was performed and the number of cross-linking sites of ABA-spermine in free 5S rRNA, or 5S rRNA incorporated into 50S subunits or 70S ribosomal complexes from E.coli ribosomes was determined. The results revealed that in free 5S rRNA spermine binds mainly to nucleotides of helices III and V and loops A, C, D and E. Integration of 5S rRNA into 50S subunits or 70S poly (U) – programmed ribosomes results in a great reduction of the susceptibility of 5S rRNA to ABA-spermine. The impact of the ABA-spermine photo-incorporation into the 5S rRNA molecule was then investigated. Chemical modification experiments revealed that, regardless of whether 5S rRNA is free or assembled in 50S subunits or 70S ribosomal complexes, binding of ABA-spermine causes significant changes in the conformation of the molecule. Loop A adopts a “looser” structure, while loops C, D, E and helices III and V achieve a more compact folding. With the discovery of changes in the configuration of 5S rRNA, due to ABA-spermine’s binding, it was interesting to study whether these conformational changes influence fundamental functions of the ribosome. Initially, the effect of changing the configuration of 5S rRNA on 50S subunit and 70S ribosomal complex assembly was investigated. The results showed that binding of ABA-spermine to 5S rRNA does not eliminate the process of 50S subunit assembly, but impedes the necessary structural changes by stabilising a more coherent structure of 5S rRNA, thermodynamically less favourable for the process. Despite the fact that binding of ABA-spermine to 5S rRNA results in structural changes that reduce the assembly of 50S subunits, when the later are formed they acquire such a structure that allows their interaction with 30S subunits. Nevertheless, the presence of 5S rRNA seems to be necessary for the formation of active 50S subunits, since 5S RNA-depleted ribosomal subunits do not associate with 30S subunits to functional 70S ribosomes. Next, the effect of photolabeling 5S rRNA was studied, initially on the ability of ribosomes to bind substrate, and then on their catalytic activity. Photolabeling of 5S rRNA with ABA-spermine caused a small but obvious activation in AcPhe-tRNA binding to the P-site. In contrast, binding to the A-site did not appear to be affected. Of great importance was the effect on the activity of peptidyl-transferase; in the presence of spermine, an increase of up to 30% on the activity of PTase was observed. Interestedly, ribosomes that lack 5S rRNA maintained ~ 10% of PTase activity, which leads to the assumption that 5S rRNA is not absolutely necessary for the formation of peptide bonds. The study of the effect of 5S rRNA conformational changes on ribosome's ability to translocate substrates followed. Spontaneous (non-enzymatic) translocation was studied first, and then, EF-G-catalyzed translocation. The results confirmed the assumption that translocation of substrates is an inherent property of the ribosome itself, even though extremely slow. At the same time, it was shown that binding of spermine to 5S rRNA, stimulates non-enzymatic translocation, possibly through changes in the folding of ribosomal RNA, that are beneficial in translocating tRNAs. Further, the presence of spermine in 5S rRNA reduced the requirements for EF-G. This result can be explained either by a possible beneficial effect of spermine to the binding of EF-G to the ribosome, either through its impact in the process of hydrolysis of GTP. To clarify which of the two versions is accurate, we studied the effect of 5S rRNA conformational changes on the ribosomal ability to bind EF-G, and then on their ability to trigger EF-G-dependent GTP hydrolysis. It was found that the presence of spermine in 5S rRNA promotes EF-G binding to ribosomes. In contrast, the results of the study showed that the initial rate of GTP hydrolysis was not influenced by the presence of spermine. Ribosomes depleted of 5S rRNA retained some GTP activity, but were unable to effectively bind EF-G even in the presence of spermine. Taking into account that EF-G binds in domain II and VI of 23S rRNA, but direct contacts of 5S rRNA with these areas or with EF-G have not been found, we are led to the conclusion that 5S rRNA affects EF-G-GTP binding to ribosomes, through allosteric signals, without affecting the EF-G-mediated GTP hydrolysis. To confirm the above, we studied the effect of the ionic environment and the changes in the tertiary structure of 5S rRNA on the conformational state of fundamental functional centres in the ribosome. Specifically, the study was focused on the ability of free spermine or photolabeling 5S rRNA with ABA-spermine to affect the SRL and GAC regions of the ribosome as well as the A-and P-sites. It was found that interaction of free spermine with ribosomes intensifies the footprint of EF-G within the ribosome and induces the AcPhe-tRNA binding to the P-site, but not to the A-site. Similar, but of lower degree, was the effect 5S rRNA labelling by ABA-spermine. In summary, a comprehensive view of the 5S rRNA nucleotide residues involved in spermine binding is gained by the present study. The results further suggest that binding of spermine to 5S rRNA causes conformational changes in certain regions of the molecule. These changes, although not of the extreme amplitude, enabled us to investigate the functional role of 5S rRNA. In light of this role, it seems that PTase center and EF-G binding center are able to coordinate their functions by transmission of allosteric signals through 5S rRNA.

The gastrointestinal (GI) tract of cats and dogs is inhabited by many different types of microorganisms, known as the GI microbiota. Mounting evidence suggests that the administration of certain dietary and/or therapeutic agents can alter the composition and activity of the GI microbiota, thus influencing gastrointestinal health and disease. The aim of this study was to evaluate the gastrointestinal microbiota in response to dietary and therapeutic interventions in cats and dogs. A multi-species synbiotic formulation, containing a total of 5x109 colony forming units of a mixture of seven probiotic bacterial strains and a blend of prebiotics, was administered daily for 21 days to healthy cats and dogs. Fecal samples were collected before, during, and up to three weeks after discontinuation of the administration of the synbiotic. The fecal microbiota was analyzed using 454-pyrosequencing, denaturing gradient gel electrophoresis, quantitative real-time PCR, and 16S rRNA gene clone libraries. The results showed that the synbiotic led to increased concentrations of probiotic bacteria in the feces but did not alter the predominant bacterial phyla. Additionally, we investigated the effect of age, body weight, and baseline abundance of probiotic related bacterial genera, as potential predictors of intestinal colonization by the ingested microorganisms. The results suggested that cats having a low abundance of fecal probiotic genera before consuming probiotics may have a higher concentration of the probiotic groups in feces during consumption of the symbiotic formulation. Also, a proton-pump inhibitor, aimed at suppressing the secretion of gastric acid, was administered daily for 15 days to healthy dogs. Changes in the GI microbiota were analyzed using 454-pyrosequencing, fluorescent in situ hybridization, and quantitative real-time PCR. The results suggested that inhibition of gastric acid secretion can alter the abundance of several gastric, duodenal, and fecal bacterial groups. However, these changes were not associated with major qualitative modifications of the overall composition of the GI microbiota. These studies showed that dietary and therapeutic agents can alter the composition of the GI microbiota and suggest that these changes could be associated with particular characteristics of the host. The clinical significance of these results needs further investigation.

Arsenic in aquifers poisons more than 100 million people in Asia alone, as aquifers remain the primary source of water for drinking and farming. Previous studies have suggested a link between the mobilisation of arsenic in aquifers and biochemical processes. As a result of the complex interaction of microbes with arsenic bearing minerals, the relatively immobile arsenate [As(V)] is reduced to labile and more soluble arsenite [As(III)] in aquifers, resulting in elevated concentrations of the metalloid. The numerous microbial communities capable of multiple-metabolic activities colonising these arsenic impacted aquifers mean that the exact mechanism of arsenic mobilisation in aquifers remains poorly understood. To resolve this ambiguity, this study undertakes a combination of metaomic, geochemical, and statistical analyses of 75 aqueous and sediment samples (three sample sets) from 3 transects with arsenic impacted aquifers in Bangladesh and Cambodia. Key geochemical and physical properties including arsenic speciation, iron speciation, mineral and elemental compositions, pH and Eh were recorded using the state-of-the art techniques of XANES, XRF, ICP-MS and other in situ techniques. Next generation sequencing (NGS) platforms such as MiSeq, HiSeq, Nextseq and Pyrosequencing, were used to sequence and analyse DNA and RNA extracted from field samples, allowing characterisation the extent bacterial communities, including any arsenic related genes and transcripts found in these arsenic impacted aquifers. The biogeochemical findings suggest that direct As redox transformations are central to arsenic fate and transport, and that there is a residual reactive pool of both As(V) and Fe(III) in deeper sediments that could be released by microbial respiration in response to hydrologic perturbation, such as increased groundwater pumping that introduces reactive organic carbon to depth. The main findings of this molecular investigation are (i) the most abundant bacterial species belonging to the families of Comamonadaceae, Moraxellaceae, Rhodocyclaceae, Gallionellaceae etc, not known for dissimilatory arsenic reduction, might possess arrA genes and thus have the potential to mobilise arsenic through dissimilatory arsenate reduction; (ii) the bacterial community structure revealed through 16S rRNA gene based sequencing and analysis, resembles the family level community structure revealed through the WGS based community analysis; (iii) although arsenic resistant genes are found in many organisms, they are transcribed only in a few organisms; (iv) the application of O2-PLS analyses may be useful for not only identifying novel organisms associated with key biogeochemical process, but also has clear potential to predict the physical/chemical environment in situ associated with microbial samples via community profiling. In conclusion, the results obtained from this study help establish the identity of microorganisms potentially playing a role in arsenic mobilisation in aquifers, and help decipher the underpinning mechanisms. This deeper level of understanding will in turn help to better target measures that can be applied to arsenic mitigation.