Philosophiae Doctor - PhD / The growth and development of apple (Malus x domestica Borkh.) fruit occurs over a period of about 150 days after anthesis to full ripeness. During this period morphological and physiological changes occur defining fruit quality. These changes are a result of spatial and temporal patterns of gene expression during fruit development as regulated by environmental, genetic and environmental-by-genetic factors. A number of previous studies partially characterised the transcriptomes of apple leaf, fruit pulp, whole fruit, and peel plus pulp tissues, using cDNA micro arrays and other PCR based technologies. These studies, however, remain limited in throughput and specificity for transcripts of low abundance. Hence, the aim of this project was to apply a high throughput technique to characterise the full mRNA transcriptome of the ‘Golden Delicious’ fruit peels and pulp tissues in order to understand the molecular mechanisms underlying the morphophysiological changes that occur during fruit development.

Les chromosomes sexuels subissent pendant la spermatogenèse de multiples modifications qui entrainent d’importantes variations dans le niveau d’expression des gènes qu’ils portent. Notamment, ils sont inactivés au cours de la méiose et la majorité reste réprimé tout au long de la spermiogenèse. Cette étude met en évidence l’existence de transcrits alternatifs particuliers de gènes sur le chromosome X et Y, dont les profils d’expressions témoignent de leur rôle au cours de ces deux phases de la spermatogenèse. Sur le chromosome X nous avons isolé, chez l’homme et chez la souris, trois gènes ubiquitaires (Uba1x, Prdx4, Atp11c) réactivés dans les spermatides via un transcrit alternatif exprimé de façon majoritaire dans les testicules. Le gène Prdx4 code, pour deux isoformes de protéines différentes par leur domaine N-terminal. Nous avons mis au point des anticorps spécifiques de chaque isoforme et nous avons démontré que, chez la souris, le transcrit réactivé est traduit dans les spermatides et produit une protéine dans un compartiment cellulaire distinct de l’autre isofome ubiquitaire. Un total de cinq mutations, affectant ces transcrits exprimés dans les spermatides, ont été retrouvées dans les gènes UBA1X et PRDX4 chez des hommes infertiles. Sur le chromosome Y chez la souris, nous avons étudié les gènes Zfy1 et Zfy2, deux homologues testicules spécifiques codant pour des protéines à doigt de zinc avec un long domaine d’activation. Zfy2, mais pas Zfy1, promeut l’élimination apoptotique des spermatocytes contenant un chromosome X univalent. Nous avons identifié un transcrit alternatif du gène testicule spécifique Zfy1 exprimé dans les spermatocytes et les spermatides. La protéine putative issue de ce transcrit, possédant un domaine acidique réduit de moitié qui pourrait être à l’origine des différences fonctionnelles entre les gènes homologues Zfy1 et Zfy2 au cours de la méiose murine. Chez l’homme, l’orthologue de ces gènes ZFY est ubiquitaire et nous avons montré qu’il produisait un transcrit alternatif testicule spécifique, codant une protéine avec le même domaine acidique raccourci que Zfy1. Nos données indiquent que ce transcrit alternatif est prédominant dans les spermatocytes et les spermatides chez l’homme et chez la souris. Ces résultats apportent la première évidence d’une fonction du gène ZFY au cours de la spermatogenèse chez l’homme et de son implication possible dans la fertilité masculine. / Sex chromosomes undergo many modifications during spermatogenesis, leading to dramatic variations in the expression levels of their genes. In particular, they are inactivated during meiosis with most genes remain silent throughout spermiogenesis. Our study describes specific alternative transcripts produced by X and Y chromosome genes, whose expression indicates roles in early spermatocytes (meiosis) and in spermatids (spermiogenesis). On the X chromosome, we have shown that three widely transcribed genes, Uba1x, Prdx4, and Atp11c, are reactivated in mouse and human spermatids via an alternative transcript that is expressed mainly in the testis. The Prdx4 gene codes two isoforms of the peroxiredoxin 4 that differ in their N-terminal domain. We have raised antibodies specific for each PRDX4 isoform and demonstrate, in mouse, that the reactivated transcript is translated in spermatids, producing a protein in a distinct cellular compartment from the ubiquitous isoform. Altogether, five mutations, affecting the spermatid-reactivated transcripts uniquely, of UBA1x and PRDX4, have been found specifically in our group of infertile men. On the mouse Y chromosome, we have studied Zfy1 and Zfy2, nearly identical testis specific zinc finger genes with long acidic (activation) domains. Zfy2, but not Zfy1, promotes the apoptotic elimination of spermatocytes with an unpaired X chromosome. We have identified an alternatively spliced transcript of Zfy1 that lacks half the acidic domain, and could explain the functional difference between Zfy1 and Zfy2. In human, the ZFY gene is widely transcribed, but we show that ZFY produces a testis specific variant transcript, encoding the same short acidic domain as Zfy1. Our data indicate that the alternative transcripts predominate in spermatocytes and spermatids, in both human and mouse. This provides the first evidence that human ZFY may play a conserved role during spermatogenesis, and contribute to human male fertility.

Antibody-mediated rejection (AMR) is the main cause of the kidney graft dysfunction and its failure after transplantation. Antibodies lead to vascular damage that is either acute or chronic and manifests as sudden or progressive graft dysfunction. Risk factors for development of AMR are time spent on haemodialysis, retransplantation, previous sensitisation against HLA antigens, and persistence of panel-reactive antibodies. Diagnosis is based on detection of deposits of C4d component of complement in peritubular capilaries and presence of donor-specific antibodies (DSA). We can also observe injury caused by antibodies against non-HLA antigens without detection of anti-HLA DSA. Use of "molecular microscope" can be beneficial in diagnosis and stratification of the risk of graft failure. High expression of ENDAT (endothelial activation and injury transcript) improves prediction of kidney graft failure more than C4d staining. Based on gene expression, the AMR scoring system correlates with the diagnosis of AMR and predicts graft loss in the future. The main goal of our work was to recognize patients at risk of AMR. In our study, we confirmed the efficacy and safety of acute AMR therapy with plasmaphereses and administration of intravenous immunoglobulins for improving outcomes of kidney transplantation....

African American Vernacular English (AAVE) has been extensively explored in previous research in sociolinguistics. However, the portrayal of the sociolect in movies is still not widely researched. In order to address this gap, the purpose of this thesis is to study how AAVE is used in the movie Sextuplets (2019), directed by Michael Tiddes and co-produced by Marlon Wayans. The material used was the script excerpted from Subslikescript (2019) [www], and the study was delimited to the speech of the two characters Alan and Dawn. The method used was close reading of these two characters’ lines in order to identify four grammatical features identified in previous research as associated with AAVE: negation ain´t+ multiple negation with ain´t, multiple negation, copula BE absence and Invariant BE. In addition, the data analysis procedure also involved identification of AAVE avoidance, i.e., instances where the characters had the opportunity to use the AAVE features but opted for their General American counterparts instead. The results show that all four AAVE features occurred in the speech of both characters, and the structures in which these features occur conforms to findings from previous studies of AAVE usage in authentic contexts. The findings also display extensive differences in frequency between the two characters’ use of AAVE. These differences can be related to their social background. Alan is portrayed as a wealthy African American male, whereas Dawn is presented as a troublemaker who has been in and out of jail. The speech of these two characters is realistic in the sense that it reproduces grammatical features of AAVE noted in previous research on language use in authentic contexts. In addition, the differences between the two characters can be said to reproduce stereotypes of how African Americans from different social classes use AAVE.

Transcriptome reconstruction is an important component in the bioinformatical part of transcriptome studies. It is particulary interesting when a reference genome is missing, highly fragmented or incomplete, since in such situations, a simple alignment (or mapping) would not necessarily tell the full story. One species with such a highly fragmented reference genome is the Norway spruce (Picea abies (L.) H. Karst.) -- a conifer, which is very important for Swedish economy. Given its long juvenile phase and irregular cone setting, the demand of cultivated seeds are larger than the supply. This yields a desire to understand the transcriptomal biology behind the cone setting in P. abies. This thesis presents an introduction to this situation, and the biological and bioinformatical background in general, followed by two papers in which this is applied: Paper I introduces a novel de novo transcriptome assembler, with a focus on recovering isoforms, and paper II makes use of this assembler to be able to detect connections between scaffolds in the P. abies genome. Paper I also studies P. abies var acrocona, a mutant with shorter juvenile phase than the wild type, in order to detect how cone setting is initiated.  From differential expression studies of both mRNA and miRNA, a number of genes potentially involved in cone-setting in P. abies were found, and also a set of miRNAs that could be involved in their regulation. / Transkriptomrekonstruktion är en viktig komponent i den bioinformatiska delen av transkriptomstudier. Särskilt intressant är detta när ett referensgenom saknas, är kraftigt fragmenterat eller ofullständigt, ty i dessa situationer skulle inte en vanlig inpassning (eller mappning) kunna berätta allt. En art med ett kraftigt fragmenterat referensgenom är gran (Picea abies (L.) H. Karst.) -- ett barrträd, som är mycket viktigt för svensk ekonomi. På grund av dess långa uppväxtsfas och oregelbundna kottsättning, så är efterfrågan av förädlade fröer större än utbudet. Detta lämnar en önskan att förstå den transkriptomala biologin bakom granens kottsättning. Denna avhandling presenterar en introduktion till denna situation, den generella biologiska och bioinformatiska bakgrunden, följd av två artiklar i vilket detta är tillämpat: Artikel I introducerar en ny de novo transkriptomassembler med fokus på att återskapa isoformer, och artikel II tillämpar denna assembler för att kunna hitta länkar mellan scaffolder (genom-delar som hittills inte kunnat länkas med varandra) i grangenomet. Artikel II studerar även granmutanten acrocona (kottegran), vilken har kortare uppväxtsfas än vildtypen, för att kunna se vad som initierar kottsättning.  Från differentiella expressionsstudier av såväl mRNA som miRNA, hittades ett antal gener potentiellt involverade i granens kottsättning, samt några miRNA som kan vara involverade i dess reglering. / <p>QC 2021-02-12</p>

The detection of non-protein-coding RNA (ncRNA) genes in bacteria and their diverse regulatory mode of action moved the experimental and bio-computational analysis of ncRNAs into the focus of attention. Regulatory ncRNA transcripts are not translated to proteins but function directly on the RNA level. These typically small RNAs have been found to be involved in diverse processes such as (post-)transcriptional regulation and modification, translation, protein translocation, protein degradation and sequestration. Bacterial ncRNAs either arise from independent primary transcripts or their mature sequence is generated via processing from a precursor. Besides these autonomous transcripts, RNA regulators (e.g. riboswitches and RNA thermometers) also form chimera with protein-coding sequences. These structured regulatory elements are encoded within the messenger RNA and directly regulate the expression of their “host” gene. The quality and completeness of genome annotation is essential for all subsequent analyses. In contrast to protein-coding genes ncRNAs lack clear statistical signals on the sequence level. Thus, sophisticated tools have been developed to automatically identify ncRNA genes. Unfortunately, these tools are not part of generic genome annotation pipelines and therefore computational searches for known ncRNA genes are the starting point of each study. Moreover, prokaryotic genome annotation lacks essential features of protein-coding genes. Many known ncRNAs regulate translation via base-pairing to the 5’ UTR (untranslated region) of mRNA transcripts. Eukaryotic 5’ UTRs have been routinely annotated by sequencing of ESTs (expressed sequence tags) for more than a decade. Only recently, experimental setups have been developed to systematically identify these elements on a genome-wide scale in prokaryotes. The first part of this thesis, describes three experimental surveys of exploratory field studies to analyze transcript organization in pathogenic bacteria. To identify ncRNAs in Pseudomonas aeruginosa we used a combination of an experimental RNomics approach and ncRNA prediction. Besides already known ncRNAs we identified and validated the expression of six novel RNA genes. Global detection of transcripts by next generation RNA sequencing techniques unraveled an unexpectedly complex transcript organization in many bacteria. These ultra high-throughput methods give us the appealing opportunity to analyze the complete RNA output of any species at once. The development of the differential RNA sequencing (dRNA-seq) approach enabled us to analyze the primary transcriptome of Helicobacter pylori and Xanthomonas campestris. For the first time we generated a comprehensive and precise transcription start site (TSS) map for both species and provide a general framework for the analysis of dRNA-seq data. Focusing on computer-aided analysis we developed new tools to annotate TSS, detect small protein-coding genes and to infer homology of newly detected transcripts. We discovered hundreds of TSS in intergenic regions, upstream of protein-coding genes, within operons and antisense to annotated genes. Analysis of 5’ UTRs (spanning from the TSS to the start codon of the adjacent protein-coding gene) revealed an unexpected size diversity ranging from zero to several hundred nucleotides. We identified and validated the expression of about 60 and about 20 ncRNA candidates in Helicobacter and Xanthomonas, respectively. Among these ncRNA candidates we found several small protein-coding genes that have previously evaded annotation in both species. We showed that the combination of dRNA-seq and computational analysis is a powerful method to examine prokaryotic transcriptomes. Experimental setups are time consuming and often combined with huge costs. Another limitation of experimental approaches is that genes which are expressed in specific developmental stages or stress conditions are likely to be missed. Bioinformatic tools build an alternative to overcome such restraints. General approaches usually depend on comparative genomic data and evolutionary signatures are used to analyze the (non-)coding potential of multiple sequence alignments. In the second part of my thesis we present our major update of the widely used ncRNA gene finder RNAz and introduce RNAcode, an efficient tool to asses local protein-coding potential of genomic regions. RNAz has been successfully used to identify structured RNA elements in all domains of life. However, our own experience and the user feedback not only demonstrated the applicability of the RNAz approach, but also helped us to identify limitations of the current implementation. Using a much larger training set and a new classification model we significantly improved the prediction accuracy of RNAz. During transcriptome analysis we repeatedly identified small protein-coding genes that have not been annotated so far. Only a few of those genes are known to date and standard proteincoding gene finding tools suffer from the lack of training data. To avoid an excess of false positive predictions, gene finding software is usually run with an arbitrary cutoff of 40-50 amino acids and therefore misses the small sized protein-coding genes. We have implemented RNAcode which is optimized for emerging applications not covered by standard protein-coding gene annotation software. In addition to complementing classical protein gene annotation, a major field of application of RNAcode is the functional classification of transcribed regions. RNA sequencing analyses are likely to falsely report transcript fragments (e.g. mRNA degradation products) as non-coding. Hence, an evaluation of the protein-coding potential of these fragments is an essential task. RNAcode reports local regions of high coding potential instead of complete protein-coding genes. A training on known protein-coding sequences is not necessary and RNAcode can therefore be applied to any species. We showed this with our analysis of the Escherichia coli genome where the current annotation could be accurately reproduced. We furthermore identified novel small protein-coding genes with RNAcode in this extensively studied genome. Using transcriptome and proteome data we found compelling evidence that several of the identified candidates are bona fide proteins. In summary, this thesis clearly demonstrates that bioinformatic methods are mandatory to analyze the huge amount of transcriptome data and to identify novel (non-)coding RNA genes. With the major update of RNAz and the implementation of RNAcode we contributed to complete the repertoire of gene finding software which will help to unearth hidden treasures of the RNA World.

Orientador: Gisela Gomes Pupo Nogueira / Banca: Giacomo Bartoloni / Banca: Sydney Molina Jr. / Resumo: Esta pesquisa constitui um estudo crítico de transcrições para violão do repertório para instrumentos de cordas dedilhadas, particularmente vihuelas, guitarras e alaúdes dos séc. XVI ao XVIII. Objetivando a proposição de subsídios teóricos para a elaboração da interpretação desse repertório, este trabalho discute os sistemas de codificação, os procedimentos de transcrição e a execução musical do repertório em questão, por meio do diálogo entre as Práticas Interpretativas e a Musicologia Histórica, assumindo como vertentes metodológicas o estudo dos sistemas de codificação, a crítica textual e a análise interpretativa de gravações. / Abstract: This research is a critical study of transcriptions for the guitar repertoire for plucked string instruments, particularly vihuela, guitars and lutes of the century. XVI to XVIII. Aiming to propose theoretical basis for the elaboration of the interpretation of this repertoire, this paper discusses the coding systems, procedures transcription and musical performance of the repertoire in question through dialogue between the practices and Historical Musicology and Interpretation Practices, assuming as methodological approaches the study of coding systems, textual criticism and the interpretative analysis of recordings. / Mestre

Η RNase P είναι το ένζυμο που ωριμάζει το 5΄ άκρο των πρόδρομων μορίων tRNA, ενώ έχει βρεθεί και στις τρεις φυλογενετικές περιοχές, καθώς και σε υποκυτταρικά οργανίδια. Είναι ριβονουκλεοπρωτεϊνικής φύσεως στις περισσότερες περιπτώσεις, ενώ έχουν βρεθεί και ένζυμα RNase P αποκλειστικά πρωτεϊνικής φύσεως. Η υπομονάδα RNA των βακτηριακών ολοενζύμων είναι καταλυτικά ενεργή απουσία πρωτεϊνικών παραγόντων in vitro, καθιστώντας την ένα πραγματικό ριβοένζυμο. Η ικανότητα της RNase P να αναγνωρίζει συγκεκριμένες δομές στα μόρια των υποστρωμάτων της και όχι αλληλουχίες, δημιούργησε τη δυνατότητα χρήσης αυτού του ενζύμου ως ενός μοριακού εργαλείου για τη στόχευση πολλών ιικών και παθολογικών μορίων RNA in vitro και in vivo, καταστέλλοντας την έκφραση των μορίων αυτών, μέσω της τεχνολογίας των μορίων EGS (external guide sequence) και των ριβοενζύμων M1GS. Η RNase P, σύμφωνα με πολλές μελέτες, έχει δειχθεί ότι αποτελεί στόχο πολλών φαρμακευτικών παραγόντων, συμπεριλαμβανομένων πολλών γνωστών αντιβιοτικών, οι οποίοι κατά κύριο λόγο αναστέλλουν τη δραστικότητα του ενζύμου. Πρόσφατα δείχτηκε, μέσω αναλυτικής κινητικής μελέτης, ότι ένα μακρολίδιο, η σπιραμυκίνη, ενεργοποιεί σημαντικά τη δραστικότητα της βακτηριακής RNase P και του M1 RNA κατά ένα δοσοεξαρτώμενο τρόπο, λειτουργώντας έτσι ως μη-ειδικός ενεργοποιητής μικτού τύπου. Μέχρι σήμερα, στη διεθνή βιβλιογραφία, δεν έχει αναφερθεί άλλη ουσία η οποία προκαλεί θετική επίδραση στη δραστικότητα της RNase P. Στην παρούσα μελέτη, αρχικά μελετήθηκε η ενεργοποίηση της δραστικότητας της βακτηριακής RNase P και αποκαλύφθηκε ότι η σπιραμυκίνη δεν αλληλεπιδρά με ιοντικούς δεσμούς με το μόριο Μ1 RNA, αλλά προκαλεί αλλαγή διαμόρφωσης στο δομικό στοιχείο P10/11 του ριβοενζύμου. Το δομικό αυτό στοιχείο εμπλέκεται στην αναγνώριση του υποστρώματος, αποτέλεσμα το οποίο έρχεται σε συμφωνία με τις τιμές KD που προσδιορίστηκαν για το σύμπλοκο ριβοενζύμου–υποστρώματος, απουσία και παρουσία σπιραμυκίνης. Με δεδομένο ότι η σπιραμυκίνη δεν επηρεάζει την πρωτεϊνοσύνθεση ή τη δραστικότητα της RNase P των ευκαρυωτικών κυττάρων, κατασκευάστηκε ένα ριβοένζυμο M1GS, ώστε να ελεγχθεί η επίδραση του αντιβιοτικού στη δραστικότητα αυτού του ριβοενζύμου in vivo, σε καλλιεργούμενα ανθρώπινα κύτταρα ΗΕΚ293. Ως στόχος του συγκεκριμένου M1GS, επιλέχτηκε ο μεταγραφικός παράγοντας Ets2 λόγω της μεγάλης κλινικής σημασίας του, εφόσον έχει συσχετιστεί με αρκετούς τύπους καρκίνου και παθολογικές καταστάσεις, καθώς και με διαδικασίες διαφοροποίησης. Ο σπουδαίος ρόλος του Ets2, σε συνδυασμό με τα ελλιπή δεδομένα σχετικά με την έκφρασή του, είχαν αποτρέψει μέχρι σήμερα την αποτελεσματική στόχευσή του με τη χρήση των υπαρχουσών μεθοδολογιών που βασίζονται στο RNA, όπως το RNAi. Μετά από ανάλυση της δευτεροταγούς δομής του Ets2 mRNA, σχεδιάστηκαν δύο οδηγοί αλληλουχίες. Οι αλληλουχίες αυτές, αρχικά, δοκιμάστηκαν ως εξωτερικές οδηγοί αλληλουχίες (EGS) σε συνδυασμό με το βακτηριακό ολοένζυμο της RNase P. Η EGS303 (το νούμερο υποδεικνύει το νουκλεοτιδικό κατάλοιπο του στόχου που δρα η RNase P), εμφάνισε τη μεγαλύτερη ικανότητα να επάγει τη δράση της RNase P in vitro. Η οδηγός αυτή αλληλουχία, στη συνέχεια κλωνοποιήθηκε στο 3΄ άκρο του M1 RNA, παράγοντας το ριβοένζυμο M1GS303, το οποίο είναι δραστικό έναντι του μορίου–στόχου του in vitro. Η δραστικότητα του συγκεκριμένου ριβοενζύμου ενεργοποιείται εντυπωσιακά κατά 160% παρουσία σπιραμυκίνης. Προκειμένου να ελεγχθεί η δραστικότητα αυτού του ριβοενζύμου in vivo, το μόριο–στόχος και το ριβοένζυμο εκφράστηκαν ελεγχόμενα σε κύτταρα E. coli, προκαλώντας μείωση της έκφρασης του μορίου–στόχου από το M1GS303 κατά 95% μετά από 12 ώρες έκφρασης των μορίων. Μείωση στα ίδια επίπεδα ανιχνεύτηκε μόλις μετά από 4 ώρες έκφρασης εφόσον στα κύτταρα είχε προστεθεί σπιραμυκίνη, γεγονός που υποστηρίζει την εντυπωσιακά θετική επίδραση της σπιραμυκίνης επί της δραστικότητας του ριβοενζύμου. Η ίδια σειρά πειραμάτων επαναλήφθηκε σε ευκαρυωτικά κύτταρα, με έκφραση του ριβοενζύμου σε HEK293 κύτταρα. Η δραστικότητα του ριβοενζύμου προσδιορίστηκε ποιοτικά και ποσοτικά, από την έκφραση της χιμαιρικής φθορίζουσας πρωτεΐνης Ets2–EGFP (μόριο–στόχος), σε διαφορετικούς χρόνους έκφρασης. Παρατηρήθηκε ότι το M1GS δρα αποτελεσματικά έναντι του μορίου–στόχου του και σε ευκαρυωτικά κύτταρα in vivo, προκαλώντας μείωση στην έκφραση του Ets2, η οποία αυξάνεται επιπλέον παρουσία σπιραμυκίνης. Τα παραπάνω αποτελέσματα δείχνουν τη σημαντική ενεργοποίηση της δραστικότητας του M1GS σε ανθρώπινα κύτταρα και καθιστούν τη σπιραμυκίνη ένα σημαντικό ενεργοποιητή στη χρήση των ριβοενζύμων M1GS ως εργαλεία γονιδιακής αποσιώπησης. Ο συνδυασμός βελτιωμένων ριβοενζύμων M1GS με την παρουσία σπιραμυκίνης αυξάνει ακόμα περισσότερο την πρακτική χρήση της συγκεκριμένης τεχνολογίας τόσο in vitro όσο και in vivo, επιτυγχάνοντας ακόμα πιο αποτελεσματική αποσιώπηση της γονιδιακής έκφρασης. / RNase P is the enzyme that endonucleolytically cleaves the precursor tRNA transcripts to produce their mature 5΄ ends. It has been found in all three phylogenetic domains of life, as well as in subcellular organelles. In most cases, it has been described as a ribonucleoprotein complex. However, few RNase P enzymes that are exclusively proteinaceous have been also reported recentrly. The RNA subunit of bacterial holoenzyme is catalytically active in the absence of protein factors in vitro, making it a true ribozyme. The ability of RNase P to recognize specific structures in its substrate molecules instead of specific sequences, allowed the use of this enzyme as a molecular tool for targeting pathological and viral RNA molecules in vitro and in vivo, by suppressing gene expression through the technology of EGS (external guide sequence) and M1GS ribozymes. RNase P, according to numerous studies, has been the target of several pharmaceutical agents, including most of the mainstream antibiotics. It has been shown recently, through analytical kinetic studies that the macrolide spiramycin significantly enhances the activity of bacterial RNase P and M1 in a dose dependent manner, acting as a non-specific mixed-type activator. Until now, no other compound has been reported to induce a positive effect on RNase P activity. In the present study, the enhancement of bacterial RNase P activity by spiramycin was tested initially, and it was revealed that spiramycin does not interact with the M1 RNA molecule through ionic bonds. On the contrary, it induces a conformational change of the P10/11 structural element of M1 RNA, which is mainly responsible for substrate recognition. The above results are in agreement with the KD values determined for the ribozyme-substrate complex, in the absence or in the presence of spiramycin. Since spiramycin does not affect eucaryotic protein synthesis or eucaryotic RNase P activity, an M1GS ribozyme was constructed, in order to examine the effect of spiramycin on the ribozyme activity in vivo, using human HEK293 cells. The target of this M1GS was the transcription factor Ets2, a factor with great clinical importance, since it has been associated with several types of cancer and disease, as well as essential processes during differentiation. The important role of Ets2 in combination with the lack of data on Ets2 expression, had hitherto prevented its effective targeting by using the existing methodologies based on RNA, such as RNAi. After analysis of the secondary structure of Ets2 mRNA, two guide sequences were designed. These sequences were originally tested in trans as external guide sequences (EGS), in combination with the bacterial RNase P. The EGS303 (the number indicates the nucleotide residue cleaved by RNase P), showed an ability to induce RNase P activity in vitro. The guide sequence was then cloned and fused into the 3' end of M1 RNA ribozyme, thus producing the M1GS303 ribozyme, which was found to be effective against the target molecule. The activity of this specific ribozyme is impressively enhanced by 160% in the presence of spiramycin. In order to examine the activity of this ribozyme in vivo, the expression of the target molecule and the ribozyme were induced in E. coli cells. After 12 hours of expression, a reduced level of the target molecule was detected, because of the M1GS303 activity (about 95%). Reduction to similar levels was observed after only 4 hours from the induction of both molecules expression, in the presence of spiramycin. This observation strongly supports spiramycin’s striking positive effect on the ribozyme activity. The same set of experiments was repeated in human HEK293 cells. The activity of the ribozyme was determined qualitatively and quantitatively, by the determination of the expression of the chimeric fluorescent protein Ets2-EGFP (target molecule) at different times of expression. The M1GS ribozyme cleaves efficiently the target molecule in human cells as well in vivo, resulting in a reduction in the expression of Ets2, which is further increased in the presence of spiramycin. This result indicates the significant activation of M1GS activity in human cells, making spiramycin an important activator in using M1GS ribozymes as tools in gene silencing. The combination of improved M1GS ribozymes in the presence of spiramycin, further increases the practical utilization of this technology both in vitro and in vivo, thus achieving an even more effective suppression in gene expression.

Proper development of skeletal muscle occurs through a highly complex process where activation and repression of genes are essential. Control of this process is regulated by timely and spatial expression of specific transcription factors (TFs). Six1 and Six4 are homeodomain TFs known to be essential for skeletal muscle development in mice. Using the C2C12 cell line, a model for skeletal muscle differentiation, I used a functional genomics approach, employing siRNA specific to both these TFs, to characterize their role in skeletal myogenesis. To identify the genes that are regulated by both these TFs, gene expression profiling by microarray of cells treated with siRNA against Six1 and/or Six4 was performed. The knock-down of these TFs caused lower expression of markers of terminal differentiation genes in addition to an impairment of myoblast fusion and differentiation. Interestingly, transcript profiling of cells treated with siRNA against myogenin revealed that several of the Six1 and Six4 target genes are also regulated by myogenin. Through a combination of bioinformatic analyses it was also found that specific knock-down of Six4 causes an up-regulation of genes involved in mitosis and the cell cycle. In summary, these results show that Six1 and Six4 can both independently regulate different genes, but can also cooperate together with other TFs where they play an important role in the proper regulation of skeletal myogenesis.

Artificial nighttime lighting (ANL) is known to adversely affect animals, but little is known what the consequences are to plants. Two genotypes of Populus balsamifera, a common urban tree, were used to investigate how ANL impacts plants. While the two genotypes varied in their physiological sensitivity to ANL, poorer levels of net leaf carbon assimilation compared to control samples suggested that ANL perturbed the perception of time of day for these plants. Gene set analysis on a subset of PopGenExpress microarray samples identified time of day specific processes in P. balsamifera, and a set of candidate ANL-sensitive genes were identified from these. Transcript measurements from the two genotypes revealed that ANL affects plants at the molecular level, for the diel cycling of the putative ANL-sensitive genes was perturbed. Together, these results suggest that ANL affects plants at the physiological and molecular level by perturbing their perception of time of day.