In this study, the novel idea of in-vivo directed evolution was applied in order to achieve variants of the enzyme galactose oxidase with increased activity. This procedure was done under starvation conditions in Escherichia coli BL21 Star (DE3). Previous studies have been carried out in order to improve the activity of this enzyme using directed evolution methods. In this study, the same idea was used in-vivo, during stationary phase adaptive mutations inside the host organism, hence called in-vivo directed evolution. This method gave variants with improved enzyme activity as compared with the wild-type enzyme, and some variants showed activities that were even higher than the variants of previous directed evolution studies, hence making this method a promising approach for the random mutagenesis of genes of interest. The above mentioned mutations are carried out by a special group of polymerases, the error-prone polymerases. Phylogenetic analysis of these error-prone polymerases was also carried out in order to investigate the relationship between the number of error-prone polymerases and the level of complexity of organisms, and both the number of error-prone polymerases and the ratio of error-prone polymerases to total DNA polymerases of six organisms were studied. It was found that as the organism gets more complex, the number of error-prone polymerases and their ratio to the total polymerases increase.

Abstract Transcription factors (TFs) play an indispensable role in cell biology as they are responsible for regulating gene activity. TFs act by binding to specific DNA sequences in a gene promoter resulting in either activation or repression of transcription of the target gene. TFs interact with target genes through a DNA binding domain which is often highly conserved and can be used to classify TFs into families. Dof (DNA binding with one finger) TFs are classified by the presence of a highly conserved bi-functional Dof domain. Characterisation of various Dof proteins has identified essential regulatory roles in plant-specific processes. This includes roles in carbon metabolism in maize, seed development and germination in Arabidopsis and cereal crops and circadian responses in Arabidopsis and rice. Despite the important role of Dof proteins in plant growth and development, this family has been studied to a limited extent in wheat with only two Dof proteins reported to date. Therefore, the aim of this thesis is to identify and initiate characterisation of the Dof TF family in wheat (Triticum aestivum) and investigate the potential role of members of this family in wheat productivity-associated physiological processes. Thirty-one Dof genes were identified after extensive searching of available Triticum aestivum ESTs and contig assembly. Phylogenetic analysis grouped these 31 genes into four clades. Extensive gene expression profiling of the TaDof family was undertaken and revealed that the majority of TaDof members were constitutively expressed in major vegetative organs with a few displaying a grain-predominant expression pattern. The TaDof family appears to be enriched with light-responsive or drought down-regulated members, suggesting that the role of this family is predominantly in growth-related processes. To further investigate their role in growth-related physiological processes, two Dof genes, TaDof4 and TaDof5, were selected for more detailed characterisation. TaDof5 was identified to be similar to the Cycling Dof Factors from Arabidopsis and the recently identified rice Dof daily fluctuations genes which are involved in the photoperiodic regulation of flowering time. TaDof5 gene expression was diurnally regulated, had strong expression in the stem and head and a peak in expression level at anthesis. Using publicly available Affymetrix data, correlation analysis suggested co-expression of TaDof5 with a number of circadian-regulated genes associated with flowering. Extensive analysis of the DNA-binding specificity of TaDof5 revealed a preferred binding motif of 5’GAAAAAGTGC. The binding of TaDof5 to DNA requires two (A/T)AAAG(T/C) core motifs in adjacent positions. The binding sequence of TaDof5 was identified in the promoter of one of the TaDof5 co-expressed genes in wheat and subsequent analysis showed that TaDof5 was capable of binding to this promoter region with high affinity. These data suggest that TaDof5 may be involved in photoperiod responses associated with flowering time. TaDof4 was among a large number of previously identified growth-related genes expressed at significantly higher levels in wheat cultivars and progeny lines with high transpiration efficiency (TE) (the amount of biomass produced per unit of water transpired). Field trials were undertaken with the parents and progeny from a cross between Quarrion (high TE) and Genaro 81 (low TE) and demonstrated that the high TE progeny lines had improved early vegetative growth. TaDof4 was therefore characterised further for a role related to biomass production and/or contribution to the TE trait. Expression profiling showed that TaDof4 was consistently expressed at higher levels in the lines with high TE, constitutively expressed in major vegetative organs, drought down-regulated and sucrose up-regulated. Over-expression of TaDof4 identified one line with significantly improved biomass. DNA binding specificity analysis demonstrated that TaDof4 binds to the AAAG(T/C) core target motif essential for Dof DNA binding. These results suggest that TaDof4 is potentially associated with growth-related processes in wheat. In conclusion this study has made the following achievements: (1) identified 31 TaDof family members in Triticum aestivum, (2) used phylogenetic analysis and expression profiling to infer potential functional roles for wheat TaDof family members, (3) further characterised TaDof5 to reveal a potential role in photoperiod responses associated with flowering and elucidated its DNA-binding specificity, (4) further characterised TaDof4 to suggest a potential role in growth-related processes. These results provide fundamental molecular information that increases our understanding of the diverse biological roles of the TaDof family, particularly in growth-related physiological processes in wheat.

The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

The purpose of this study is to characterise Australian isolates of bovine enterovirus (BEV) and develop a suitable isolate as a replication-limited vaccine vector. Advantages of using BEV as a vector are that it both elicits mucosal immunity and has naturally occurring temperature stable isolates so that a BEV vector could be administered orally to elicit a protective immune response in the host and should not require cold storage to maintain vaccine efficacy. Furthermore, wildtype BEV causes no or only mild clinical symptoms in its host and if BEV is used as a vaccine vector, reversion to wildtype phenotype would not cause deleterious effects in vaccinated cattle. To date many of the viruses used as vaccine vectors are produced by modifying the structural proteins of the virion so that they contain heterologous sequences. However, each of the four BEV structural proteins are essential and it is not possible to insert large sequences without disrupting the virion. While this study looks at potential insertion sites within the BEV virion, the main focus for the development of BEV as a vaccine vector is through using a replication-limited BEV vector. The development of a replication-limited vector requires the deletion of an essential viral gene that is then replaced in vitro using an expression vector. When the replication-limited vector and its complementing expression cassette are co-transfected into a permissive cell line all the proteins required for viral assembly are produced but only replication deficient genomes are available to be encapsidated. The physically intact but replication deficient viral particles produced in vitro can then infect permissive cells in vivo, resulting in the production of all but the deleted viral protein. Moreover, the deleted portion of the viral genome can be replaced with heterologous sequences within the replication-limited BEV vector. These heterologous sequences can then be expressed in vivo where they can be recognised by the host immune system. Three BEV isolates representing the Australian subserotypes were used in this study: K2577, SL305 and 66/27. The full-length sequences of K2577 and SL305 were obtained as well as partial sequence from the third isolate, 66/27. Sequence homology and phylogenetic analysis showed all three isolates were more closely related to BEV-1 subserotypes than BEV-2. This is the first report to indicate that Australian BEV isolates can be classified as BEV-1. Analysis of the 5’-untranslated region (5’-UTR) indicated that BEV isolates were recombinants with each other and that these recombinant regions correspond to the duplicated cloverleaf structure which is present in BEV 5’-UTR but absent from other enteroviruses. While BEV was initially reported to be stable at higher temperatures, later studies showed that this property varied between isolates and this is also true of the three isolates used in this study. Since it is important not only to ensure that the isolate used as a vaccine vector is temperature stable but also the resulting vaccine vector, the molecular basis of BEV temperature stability was also studied. Using sequence data from the Australian isolates, regions of variation were located and hybrid BEV created. Unfortunately, all of the hybrid BEV produced in this study were non-infectious and could not be used to for further characterisation of BEV temperature stability. Preparatory to constructing replication-limited BEV, a system for full-length amplification of BEV was developed. By including sequences for the bacterial promoter T7 on the positive sense primer used for full-length amplification of BEV, it was possible to prepare full-length transcripts of the amplified product and these were shown to produce infectious BEV particles when transfected into to cell lines that supported BEV growth. Subsequent cloning of the K2577 amplification product resulted in infectious clones for this BEV isolate and these clones were used to prepare replication-limited BEV constructs. To test the replication-limited system BEV structural genes were replaced with a reporter gene to produce replication deficient infectious clones. Complementary constructs containing only the deleted structural genes were also prepared to express the deleted genes. While it was expected that these expression vector would be able to complement the replication deficient BEV in vivo, co-transfection of the replication-limited construct with its complementing expression vector did not produce viable BEV.

Στο πλαίσιο της παρούσης διατριβής, αξιοποιήθηκαν οι αλληλουχίες γνωστών γονιδιωμάτων, αλλά και γονιδιωμάτων που αποκρυπτογραφήθηκαν πρόσφατα, για να μελετηθεί η εξελικτική ιστορία τριών λειτουργικά σημαντικών πρωτεϊνικών οικογενειών: (α) των φυτικών DNA μεθυλομεταφορασών και (β) των ευκαρυωτικών RNA μεθυλομεταφορασών, οι οποίες είναι ένζυμα που επιφέρουν μεθυλίωση στις νουκλεοτιδικές αλληλουχίες, καθώς και (γ) των πεπτιδασών συγγενών της καλλικρεΐνης (KLKs), οι οποίες είναι γνωστές σερινοπρωτεϊνάσες με δράση τύπου θρυψίνης ή χυμοθρυψίνης. Οι εξελικτικές σχέσεις των χαρακτηρισμένων και καινοφανών πρωτεϊνών των τριών οικογενειών διερευνήθηκαν με κατασκευή φυλογενετικών δένδρων. Επίσης, αναλύθηκε η δευτεροταγής και τριτοταγής δομή των ομόλογων πρωτεϊνών, η δομή των γονιδίων που κωδικοποιούν τις πρωτεΐνες αυτές, και κατασκευάσθηκαν διαγνωστικά πρωτεϊνικά μοτίβα από τις αλληλουχίες των τριών οικογενειών ενζύμων. Τα αποτελέσματα των αναλύσεων οδήγησαν στην εξαγωγή σημαντικών συμπερασμάτων σχετικά με την πιθανή βιολογική λειτουργία των καινοφανών πρωτεϊνών. Συγκεκριμένα, ομόλογες φυτικές DNA μεθυλομεταφοράσες και καινοφανείς ευκαρυωτικές RNA μεθυλομεταφοράσες ταυτοποιήθηκαν σε δημόσιες βάσεις δεδομένων. Λεπτομερής φυλογενετική ανάλυση οδήγησε στην ταυτοποίηση των τεσσάρων ήδη γνωστών οικογενειών φυτικών DNA μεθυλομεταφορασών και μιας καινοφανούς υποοικογένειας (Pavlopoulou and Kossida, 2007). Επίσης, ταυτοποιήθηκαν πέντε υποοικογένειες ευκαρυωτικών RNA μεθυλομεταφορασών. Πέραν των τριών ήδη γνωστών υποοικογενειών (NOP2, NCL1 και YNL022C), ταυτοποιήθηκε μια καινοφανής υποοικογένεια (RCMT9), και μια υποοικογένεια, FMU, η οποία εθεωρείτο ότι απαντάται αποκλειστικά σε προκαρυωτικούς οργανισμούς (Pavlopoulou and Kossida, 2009). Επιπλέον, κατασκευάσθηκαν πρωτεϊνικά αποτυπώματα της οικογένειας (και των επιμέρους υποοικογενειών) των RNA μεθυλομεταφορασών, τα οποία καταχωρήθηκαν στη δευτερογενή πρωτεϊνική βάση δεδομένων PRINTS (http://www.bioinf.man.ac.uk/dbbrowser/PRINTS). Στα πλαίσια της διατριβής, αναπτύχθηκε το υπολογιστικό πρόγραμμα RCMTHMM, με σκοπό τη διάκριση/ταυτοποίηση των ευκαρυωτικών RNA μεθυλομεταφορασών, το οποίο διατέθηκε για δημόσια χρήση στη διεύθυνση URL: http://www.bioacademy.gr/bioinformatics/RCMTHMM. Σημαντική συνεισφορά της παρούσας μελέτης είναι η αναδιατύπωση της εξελικτικής ιστορίας των καλλικρεϊνών (Pavlopoulou et al., 2010). Οι καλλικρεΐνες είναι σημαντικά πρωτεολυτικά ένζυμα που δρουν ατομικά ή σε πρωτεολυτικούς καταρράκτες και ρυθμίζουν σημαντικές φυσιολογικές λειτουργίες, ενώ η απορρυθμισμένη δράση τους έχει συνδεθεί με σοβαρές ασθένειες (καρδιοαγγειακές, νευροεκφυλιστικές, φλεγμονώδεις, δερματικές, διάφορους τύπους καρκίνου). Για την απομόνωση νέων καλλικρεϊνών αξιοποιήθηκε το γεγονός ότι τα KLK γονίδια συνεντοπίζονται στο ίδιο χρωμόσωμα υπό μορφή μη διακοπτόμενης συστάδας γονιδίων. Σε προηγούμενες μελέτες είχε προταθεί ότι οι καλλικρεΐνες απαντώνται μόνον στα θηλαστικά και ότι εμφανίσθηκαν πριν από περίπου 150 εκατομμύρια χρόνια. Στην παρούσα μελέτη, ομόλογες καινοφανείς ακολουθίες καλλικρεϊνών ανιχνεύθηκαν in silico στα γονιδιώματα διάφορων οργανισμών. Ορθόλογα των καλλικρεϊνών ταυτοποιήθηκαν για πρώτη φορά στα ερπετά, στα πτηνά και στα αμφίβια, υποδεικνύοντας την εξελικτική καταγωγή των καλλικρεϊνών πριν από 330 εκατομμύρια χρόνια. Επιπροσθέτως, πέραν των 15 γνωστών KLKs (KLΚ1-15), ταυτοποιήθηκαν τρία καινοφανή μέλη (ορφανές Klks) και με σύγκριση των προβλεπόμενων δομών δείχθηκε ότι τα δομικά χαρακτηριστικά που σχετίζονται με την καταλυτική δράση είναι συντηρημένα στις καινοφανείς πρωτεϊνικές αλληλουχίες KLK. Σημειωτέον, δείχθηκε ότι στα γονιδιώματα όλων των υπό εξέταση οργανισμών, τα ορθόλογα γονίδια KLK χαρτογραφούνται στην ίδια χρωμοσωμική περιοχή, διευθετημένα εν σειρά με τον ίδιο προσανατολισμό και χωρίς παρεμβολή μη καλλικρεϊνικών γονιδίωνΠροτείναμε ότι η οικογένεια των καλλικρεϊνών προέκυψε από μια σειρά γονιδιακών διπλασιασμών και μεταλλάξεων, και ότι οι καλλικρεΐνες έχουν συνεξελιχθεί με τα ειδικά υποστρώματά τους (Pavlopoulou et al., 2010). / In the present thesis, the availability of an increasing number of complete or almost complete genomes, including those that were completed recently, enabled the study of the evolutionary history of three functionally important protein families: (a) the plant DNA methyltransferases and (b) the eukaryotic RNA methyltransferases, which are enzymes that catalyze the transfer of a methyl group to nucleotide sequences, as well as (c) the kallikrein-related peptidases or KLKs, which are trypsin- or chymotrypsin-like serine proteases. The evolutionary relationships of the already known and the novel proteins of the three families that were identified here were investigated using phylogenetic trees. Moreover, the secondary and tertiary structures of the homologous proteins were analyzed, as well as the structure of the protein-encoding genes, and diagnostic protein motifs were constructed based on the sequences of the three enzyme families. Our results led to suggestions pertaining to the biological function of the identified novel proteins. In particular, homologous plant DNA methyltransferases and novel eukaryotic RNA methyltransferases were identified in publicly accessible sequence databases. Detailed phylogenetic analysis of plant DNA methyltransferases identified four already known families and a novel subfamily in addition (Pavlopoulou and Kossida, 2007). Moreover, five distinct eukaryotic RNA methyltransferase subfamilies were identified; apart from the three already known subfamilies (NOP2, NCL1 and YNL022C), one novel subfamily (RCMT9) and the FMU which hitherto was considered to exist exclusively in prokaryotes were also identified (Pavlopoulou and Kossida, 2009). Furthermore, protein fingerprints were constructed from the generic family of RNA methyltransferases (and the individual subfamilies), which were deposited in the PRINTS database (http://www.bioinf.man.ac.uk/dbbrowser/PRINTS). We developed the computational program RCMTHMM, in order to discriminate/identify eukaryotic RNA methyltransferases from other proteins. The RCMTHMM program has been made publicly available in the URL: http://www.bioacademy.gr/bioinformatics/RCMTHMM. Finally, the evolutionary history of KLKs was reconstructed. Kallikreins are important proteolytic enzymes which are involved in proteolytic cascade pathways and their dysregulated expression has been associated with major human pathologies (cardiovascular diseases, neurodegenerative disorders, inflammatory diseases, skin diseases, different cancer types). The prominent feature of the kallikrein family is that it consists of tandemly and uninterruptedly arrayed genes on a single locus at human chromosome 19q13.3-13.4. This unique feature was used in order to identify novel KLKs and KLK-like genes/proteins. Previous studies on the evolution of kallikreins were restricted to mammals and the emergence of the kallikrein genes was suggested approximately 150 million years ago. In the present study, homologous novel kallikrein protein sequences were detected in silico in the genomes of various species. For the first time, novel KLK orthologues were identified in reptiles, aves and amphibia, which allowed us to trace the evolutionary origin of kallikreins 330 million years ago. In addition, apart from the 15 already known KLK genes (KLK1-15), three novel members were identified (orphan Klks). All the defining structural features which are related to the catalytic activity of KLKs were found to be conserved in the novel KLK protein sequences. Of particular interest, the synteny of the KLK-encoding genes was analyzed and it was shown that these genes are co-localized in contiguous, uninterrupted clusters maintaining the same orientation in all species under investigation. We suggest that a series of gene duplication and mutation events gave rise to the family of KLK enzymes and KLKs have co-evolved with their specific substrates (Pavlopoulou et al., 2010).

RESUMO Alfonso Castro, H. L. Caracterização molecular de dengue tipo 3 isolados no Brasil e no Paraguai. 2010. 105f. Dissertação (Mestrado). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2010. O vírus da dengue (DENV), pertencente ao gênero Flavivirus da família Flaviviridae, é a arbovirose de maior impacto em saúde pública na atualidade. A infecção com qualquer do quatro sorotipos de dengue (DENV-1, -2, -3 e -4) pode ser assintomática ou causar doença febril (DF) que pode evoluir para uma forma mais grave, e algumas vezes fatais, caracterizada por derrame capilar, trombocitopenia. A introdução do DENV-3, genótipo III, nas Américas coincidiu com um aumento no número de casos graves da doença. Este vírus causou uma grande epidemia em 2002 no Rio de Janeiro e posteriormente se espalho em todas as regiões do pais, chegando inclusiva ao Paraguai. Diversos estudos filogenéticos e evolutivos foram realizados com o DENV-3 nas Américas, mas utilizando sequências genômicas parciais. Neste trabalho temos por objetivo analisar o relacionamento filogenético e evolutivo de DENV-3 isolados no Brasil e no Paraguai analisando a sequência genômica completa. A sequência de vírus isolados no Brasil (n=9) e no Paraguai (n=3) foram comparadas com 527 sequências depositadas no GenBank. As 12 cepas virais isoladas no Brasil e no Paraguai pertencem ao grupo americano do genótipo III. Analisando a árvore filogenética dos DENV-3 observamos três genótipos e diversas linhagens, sub-linhagens e clados dentro de cada genótipo. A distância genética entre os genótipos foi de 7,3 a 7,5%, entre as linhagens de 3,2 a 5,3%, entre as sub-linhagens 2,5 a 3,2% e entre os clados de 1,0 a 1,9%. A taxa evolutiva dos vírus variou entre 1,2x10-4 a 8,2x10-4 subs/sitio/ano. O ancestral comum do genótipo I teria surgido entre 1849-1945, do genótipo II entre 1916-1960, e do genótipo III entre 1876-1923. Os diferentes grupos genéticos apresentam motif de aminoácidos característicos. Estes dados serão de grande utilidade para uma melhor caracterização dos DENV-3 em futuras epidemias e, inclusive, poderão ser utilizados para seleção de candidatos a vacina. / ALFONSO CASTRO, H. L. Molecular characterization of dengue type 3 isolated in Brazilian and Paraguay. 2010. 105f. Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto Universidade de São Paulo, Ribeirão Preto, 2010. Infections of humans with dengue viruses (DENV), which belong to the genus Flavivirus(family, Flaviviridae), can be subclinical or cause illnesses ranging from a mild, flu-like syndrome with rash (dengue fever [DF]) to a severe and some times fatal disease, characterized by capillary leakage, thrombocytopenia, and sometimes hypovolemic shock (hemorrhagic dengue fever [DHF/DSS]). DENV are classified in four immunological distinct serotypes: DENV-1 to 4. Recently, a dramatically increase of DHF/DSS cases in the Americas have bee see, and this increase coincided with the introduction of the dengue virus type 3, genotype III. This virus causes a great epidemic in 2002 in the city of Rio de Janeiro and later, the virus spread in Paraguay. Phylogenetics and evolutionary studies have bee carried out with DENV-3 isolated worldwide, but using sequences partial genomic. In this work, we have analyzed the genetic diversity of DENV-3 of Brazilian and Paraguayan isolated, analyzing the complete sequences genomic. The Brazilian (n=9) and Paraguayan (n=3) isolated, were compared with 527 sequences deposited in the GeneBank. Theses isolated, belong to the American group of the genotype III. The phylogenetic analysis of complete genome of the DENV-3, confirmed the existence of three known genotypes and suggested the presence of other groups within each genotype named of the lineages, sub-lineages and clades. The genetic distance among the genotypes were of 7,3 to 7,5%, among the lineages of 3,2 to 5,3%, among the sub-lineages of 2,5 to 3,2% and among clades of 1,0 to 1,9%. The evolutionary rates of the viruses varied among 1,2x10-4 to 8,2x10-4 s/s/y. The age of the ancestral common more recent of the genotype I, possibly are among 1849-1945, the ancestral common of the genotype II, among 1916-1960 and the ancestral common more recent of the genotype III, among 1876-1923. The different genetic groups present motif of amino acids. These data could provide information for a better understanding of the evolution of theses viruses, and even for selection of candidate vaccine

Made available in DSpace on 2015-04-17T14:55:26Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 8341348 bytes, checksum: 3c793b10a08a57a4fe65df46346e7385 (MD5) Previous issue date: 2013-03-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The first classifications for the annelids were presented within a peculiar group of worms grouped within Class Vermes. The group was initially divided into Errant Annelides, Tubicolous or Sedentary Annelides, Terricolous Annelids, and Freshwater Annelids. These classifications did not reflect common ancestry. With the advent of phylogenetic systematics, many proposals were made for other organisms, attempting to reflect true relationships. The first proposals for annelids and polychaetes appeared in the 90s, based on morphology, and attempted to confirm the monophyly of these two groups. In these analyses, the Pogonophora were reduced to a family of Polychaeta, the Siboglinidae. These results remained incongruent when compared to results obtained later from molecular data. Another phylogenetic proposal presented the Pogonophora as being close to the sedentary polychaetes, closely related to Owenia. In this proposal, the clade Metameria was established to group the annelids, Enterocoela and Deuterostomia. Pogonophora as a family of Polychaeta disregards the evolutionary relationships that this taxon shares with the deuterostomes. In the present work, polychaetes with tori were selected as the ingroups of the analysis, together with Pogonophora, and including Phoronida and Pterobranchia, in order to establish genealogical relationships among these taxa. For parsimony analyses molecular data from 18S rRNA, morphological data coded as binary (a/p), multistate, and combined data (multistate molecular and morphological data) were used. Several slightly different topologies appeared in our results on morphology and molecules. On the other hand, the combined data was similar to the topology obtained from multistate morphology. From these analyses, we hypothesize that sedentary polychaetes with tori (including Pogonophora) are strictly related to Phoronida and Deuterostomia, their tagmatization being considered a particularly important synapomorphy. Finally, we emphasize the paraphyletic nature of Protostomia, Spiralia, Trochozoa and Lophotrochozoa, which are contrasted to the monophyletic Metameria. / As primeiras classificações para os anelídeos foram representadas para um grupo peculiar de vermes que formavam as primeiras famílias de poliquetas, agrupadas dentro da Classe Vermes. O grupo foi dividido inicialmente em Annélides Errantes, Annélides tubicoles ou Sédentaires, Annélides Terricoles e Annélides souceuses. Essas classificações não refletiam ancestralidade comum. Com o surgimento da sistemática filogenética, muitas propostas foram apresentadas para vários outros grupos de organismos, buscando refletir as relações de parentescos. A partir da década de 90 surgiram os primeiros trabalhos de filogenia com dados morfológicos para os anelídeos e poliquetas, com objetivo de confirmar a monofila dos dois grupos. Nestas análises, Pogonophora foi reduzido a uma família de Polychaeta, os Siboglinidae. Os resultados permaneceram incongruentes quando comparados os dados morfológicos com os dados moleculares, que surgiram posteriormente. Outras propostas filogenéticas apresentaram os Pogonophora como grupo próximo aos poliquetas sedentários, relacionados com os Owenia. Nessa proposta, foi estabelecido o clado Metameria para agrupar anelídeos, Enterocoela e Deuterostomia. Pogonophora como uma família de Polychaeta quebra a relação de paradigma evolutivo que este táxon compartilha com os Deuterostômios. Neste trabalho, se usou como grupo interno poliquetas com tori, Pogonophora, Phoronida e Pterobranchia, a fim de estabelecer relações genealógicas entre eles. Desta forma, se usou para análise de parcimônia dados moleculares 18S rRNA, dados morfológicos codificados como binário e multiestados, e dados combinados (moleculares e morfológicos multiestados). Os resultados mostraram várias hipóteses que se diferenciaram um pouco nas topologias, quando foram comparados os cladogramas de caracteres moleculares com os cladogramas de caracteres morfológicos. Embora, a topologia de caracteres combinadas se mostrou igual à topologia de caracteres morfológicos multiestados. Dessa maneira, hipotetiza-se a partir das análises aqui obtidas, que os poliquetas sedentários portadores de tori (incluindo Pogonophora) estão estritamente relacionados aos Phoronida e Deuterostomia, principalmente quando se ressalta o processo de tagmatização. Finalmente, enaltece-se a parafilia de Protostomia, Spiralia, Trochozoa e Lophotrochozoa, ressaltando o monofiletismo de Metameria.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-09Bitstream added on 2014-06-13T18:09:03Z : No. of bitstreams: 1 martins_c_me_rcla.pdf: 494549 bytes, checksum: 956ee49fb3cabf6b959eecbb86566527 (MD5) / O gênero Solenopsis tem distribuição cosmopolita, mas espécies do grupo S. saevissima, são nativas da América do Sul e inclui as conhecidas formigas lava-pés. Elas foram introduzidas de forma acidental em diversas regiões biogeográficas do mundo. No Brasil possuem ampla distribuição, mas têm preferência por áreas de atividade humana. São formigas altamente agressivas e são responsáveis por acidentes que podem levar a choques anafiláticos e à morte. As formigas apresentam associações de diferentes tipos com outros organismos, inclusive com bactérias endossimbiontes como a Wolbachia, bactéria intracelular que também infecta as formigas do gênero Solenopsis. No presente trabalho, procurou-se caracterizar as populações de lava-pés (Solenopsis spp.) de ampla área do território brasileiro, analisando o parentesco dessas populações e inferindo sobre sua filogenia. Além disso, foi investigada a presença, frequência e distribuição do endossimbionte Wolbachia em populações de Solenopsis spp. no Brasil. A caracterização das lava-pés foi baseada na análise do gene citocromo oxidase I e em estudos filogenéticos. Observou-se desde completa coerência geográfica, até polifilia para as espécies S. invicta e S. saevissima, o que demonstra claramente a diversidade desse gênero de formigas no Brasil. Existe a possibilidade de ocorrer populações isoladas reprodutivamente, tendo como decorrência processos evolutivos de especiação. Além disso, clados com espécies divergentes agrupadas podem trazer evidências de espécies erroneamente identificadas morfologicamente, presente em bancos de dados. O levantamento da ocorrência de Wolbachia foi baseado no gene wsp do endossimbionte e análises filogenéticas foram realizadas para inferir a história evolutiva dessas bactérias nas populações de lava-pés do país. Foi encontrada uma grande... / The genus Solenopsis has a cosmopolitan distribution, but species of S. saevissima group are native from South America and include the known fire ant. They were accidentally introduced in several countries of the world. In Brazil they have wide distribution with preference for areas of human activity. Ants are highly aggressive and responsible for accidents that can lead to anaphylactic shock and death. The ants have different associations with other organisms, including bacteria endosymbionts such as Wolbachia, intracellular bacteria that also infect the ants of the Solenopsis genus. In this study, we sought to characterize the populations of fire ants (Solenopsis spp.) in a wide area of Brazil, analyzing the relationship of these populations and inferring their phylogeny. Furthermore, we investigated the presence, frequency and distribution of the endosymbiont Wolbachia in those populations of Solenopsis spp. in Brazil. The characterization of fire ants was based on analysis of the cytochrome oxidase I gene and on phylogenetic studies. It was observed that there were complete geographical coherence and polyphyly for the species S. invicta and S.saevissima, which clearly demonstrate the diversity of this genus of ants in Brazil. There is the possibility to occur reproductively isolated populations, leading to evolutionary processes of speciation. Furthermore, clustered clades with divergent species can bring evidences of species wrong morphologically identified, presents in databases. The survey of the occurrence of Wolbachia was based on the wsp gene of the endosymbiont and the phylogenetic analyses were performed to infer the evolutionary history of these bacteria in the populations of fire ants. There was a great diversity of Wolbachia in the genus Solenopsis, with 51% of the analyzed colonies presenting infection and the highest incidence was found in populations from... (Complete abstract click electronic access below)

Made available in DSpace on 2015-04-01T14:16:04Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1756619 bytes, checksum: d052f1be18e53f2beddaa57cda5b0a22 (MD5) Previous issue date: 2012-12-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Substrate-binding subunits are very important components of the solute importation system, known as the osmoprotectant system, which consists of a membrane protein belonging to the ABC superfamily. These molecules recognize specific substrates that have different physiological roles in prokaryotes, i.e. roles that contribute to the survival of these organisms in environments with high concentrations of salt. Using MEGA 5.05 software, this study performed a phylogenetic analysis of 431 nucleotide sequences of these subunits, orthologous to each other, collected from the database contained on the website http://www.genome.jp/kegg/. As a result of this analysis, phylogenetic trees were generated that clearly demonstrated that there was a horizontal transfer of some genes due to the sharing by different organisms. Also, two probable ancestral sequences were generated that showed homology with permeases that transport choline, glycine betaine and carnitine, which are trimethylamines currently present in various prokaryotes. Therefore, this system probably arose in prokaryotic organisms with the basic function of capturing nutrients, and by performing this basal function of being shared with other organisms, was fixed to the genome. However, because of the diversification of habitats by the prokaryotes, this system contributed decisively to the adaptation of these organisms to different environments, especially environments that had a high salt concentration; thus, acting and being currently characterized as a system of osmoprotection. / As subunidades ligadoras de substrato são componentes muito importantes do sistema de importação de soluto conhecido, como sistema de osmoproteção, que consiste em uma proteína de membrana pertencente à superfamlía ABC. Estas moléculas reconhecem substratos específicos que apresentam papéis fisiológicos diversos em procariotos, incluindo a colaboração para a sobrevivência destes organismos em ambientes com elevada concentração de sal. Utilizando o software MEGA 5.05, foi realizada uma análise filogenética de 431 sequências nucleotídicas destas subunidades, ortólogas entre si, coletadas a partir do banco de dados contido no site ttp://www.genome.jp/kegg/. Como resultado desta análise, foram geradas árvores filogenéticas que demonstraram claramente que houve a transferência horizontal de alguns dos genes devido ao ompartilhamento por organismos diferentes. Foram geradas também duas prováveis sequências ancestrais que apresentam homologia com permeases que transportam colina, glicina betaína e carnitina, que são aminas trimetiladas, presentes atualmente em diversos procariotos. Portanto, este sistema provavelmente surgiu em organismos procarióticos com a função básica de captura de nutrientes e por desempenhar esta função basal, ao ser compartilhado com outros organismos, foi fixado aos genomas. No entanto, a partir da diversificação de habitats, por parte dos procariotos, este sistema colaborou de forma decisiva para a adaptação destes organismos aos mais diversos ambientes, incluindo, especialmente os ambientes que apresentavam uma elevada concentração de sal, atuando e sendo caracterizado atualmente como um sistema de osmoproteção.