The genes that regulate pubertal timing in the general population are not well understood. Recently, genome-wide association studies have demonstrated that genetic variants near LIN28B associate with variation in pubertal timing in humans. To investigate where within the hypothalamic-pituitary-ovarian (HPO) axis Lin28b, and its homologue Lin28a, regulate pubertal timing, expression of these genes was assessed across the pubertal transition. The finding that Lin28a/b expression decreases only in the ovary suggests that the Lin28 pathway may exert its regulatory effects with respect to puberty in the ovary. Another aim of this thesis was to examine the effect of estrogen on Lin28b expression in immortalized GnRH neuronal cells, but the data remains equivocal and detailed future studies are needed to make definitive conclusions. The ovarian expression data lay the foundation for further studies using conditional knockout mice to verify the importance of the tissue and age specific developmental pattern that was identified.

Members of the important disease causing bacterial generas, Chlamydia and Chlamydophila, are characterised by a complex developmental cycle which is comprehensively described by microscopy. The inability to use standard genetic techniques for this obligate intracellular bacterium, however, means that significant gaps in our understanding of the molecular mechanisms used to control growth and development of Chlamydia still exist. The current study investigated the function of bacterial guanosine triphosphatases (GTPases), components of the organism's limited signal transduction arsenal, in regulatory control of the chlamydial development cycle. Initial analysis of the gene transcription of chlamydial GTPases and other predicted signal transduction genes using real time RT-PCR, in a Chlamydophila pneumoniae A-03 tryptophan depletion model of persistence, revealed significant differential expression of genes in response to the addition of interferon gamma (IFN-γ). Predicted chlamydial GTPase encoding genes, ychF, yhbZ and yphC, associated with ribosome function amongst other processes were strongly up-regulated, while hflX was down-regulated in the persistent cultures. Analysis of an additional model of Cp. pneumoniae persistence, induced by limitation of host cell iron, revealed that ychF, yhbZ and yphC were also up-regulated in the persistent cultures. This study provided the most comprehensive analysis of Cp. pneumoniae gene transcription to date and suggest that chlamydial GTPases serve a role in generation of the persistent chlamydial phenotype. Cloning and expression of Cp. pneumoniae and Cp. abortus yhbZ, including demonstration of in vitro GTPase activity, indicates that this chlamydial gene encodes a member of the universally conserved and essential bacterial Obg subfamily of GTPases. Evidence is building that members of this latter family of bacterial GTPases are important regulators of bacterial growth and morphological differentiation in developmentally complex bacteria. Over-expression of chlamydial YhbZ subfamily GTPases in Escherichia coli revealed inhibition of bacterial growth and disruption of cell division and chromosome functions leading to the generation of elongated cells with limited chromosome segregation, as described for Obg subfamily members from E. coli and other bacteria. Although more analysis is required, we suggest a novel mechanism of chlamydial Obg GTPase regulation involving sensing of host cell GTP/GDP pools to control secondary differentiation of reticulate bodies (RBs) back to elementary bodies (EBs). Analysis of the chlamydial complement of bacterial GTPases was extended to HflX, a previously uncharacterised and only predicted GTPase conserved in bacteria. HflX sequence analysis revealed conservation of G motifs responsible for nucleotide binding and hydrolysis (G1, G3, G4) and protein interaction (G2), although the latter was unique to HflX subfamily GTPases. Recombinant Cp. pneumoniae HflX displays GTPase activity with nucleotide specificity for GTP. We tested Cp. pneumoniae HflX function by over-expression in E. coli which led to inhibition of growth in E. coli and elongation of cells with normal chromosome partitioning. This phenotype was the probable result of disruption of a stage in cell division subsequent to chromosome segregation. This present study provides the first evidence to show that bacterial HflX is a GTPase and suggests a regulatory role in bacterial cell cycle control.

Chlamydial infections have been associated with several chronic human diseases, including trachoma, pelvic inflammatory disease, chronic obstructive pulmonary disease and atherosclerotic cardiovascular disease. In Chlamydia-associated disease, the organisms are believed to exist in an atypical, persistent phase that is not well understood at the genetic level. The research presented in this thesis investigated chlamydial gene expression in in vitro cell culture models of persistence. The first set of studies analysed a continuous-infection model of persistence that has been recently developed for two C. pneumoniae isolates (TW-183 and CM-1). The spontaneous establishment and unique cyclical nature of continuous infections could be particularly relevant to in vivo events. An initial analysis using a semi-quantitative reverse transcriptase PCR (sqRT-PCR) approach provided evidence of differential gene expression in C. pneumoniae TW-183 continuous infections relative to acute control infections. Using a subsequently established fully quantitative real-time reverse transcriptase PCR (rtRT-PCR) assay, up-regulated expression profiles were confirmed for five genes (CPn0483, nlpD, ompA, pmp1 and porB) in the continuous C. pneumoniae TW-183 infections. The omcB, pmp1 and porB genes, all of which encode membrane proteins, showed similar patterns of expression over both the acute and continuous time courses tested. Gene expression data for a second C. pneumoniae isolate, CM-1, revealed similar overall expression trends to those seen for C. pneumoniae TW-183 but also supported previous observations of different growth characteristics between the two isolates in the continuous-infection model. The rtRT-PCR assay was further optimised for use in gene expression studies of the gamma interferon (IFN-γ)-mediated model of C. pneumoniae A-03 persistence, in which altered growth and morphological traits typical of chlamydial persistence have been well characterised. Meanwhile, chlamydial genes such as euo, ftsK and hctB were emerging from the literature as reliable genetic markers of persistence. Therefore, a preliminary rtRT-PCR analysis of marker gene expression was used to assess the likely extent of persistence in individual IFN-γ-treated C. pneumoniae A-03 infections from a series of experiments that had been prepared for this persistence model. In this way, an appropriate pair of duplicate experiments was selected for further studies based on strong genetic evidence of persistence in IFN-γ-treated samples at 48 h post-infection (PI) in those experiments. Using rtRT-PCR, 14 genes of interest from the related peptidoglycan, aminosugars and lipopolysaccharide (LPS) biosynthetic pathways were analysed in the validated experiments of the IFN-γ-mediated C. pneumoniae A-03 persistence model. Selective up- and down-regulated expression trends were associated with IFN-γ-treatment at 48 h PI for genes encoding products that are located at specific enzymatic points in these pathways. Most strikingly, the expression of glmU, the product of which controls the amount of an essential precursor metabolite that enters both peptidoglycan and LPS biosynthesis, was strongly and reproducibly down-regulated in the 48-h PI IFN-γ-treated samples. This expression profile may contribute to a reduced rate of peptidoglycan biosynthesis in this persistence model and may therefore be related to the inhibited cell division and RB-to-EB differentiation that characterise chlamydial persistence. While most other genes in these pathways showed unchanged expression associated with IFN-γ treatment, murA and kdsB (from peptidoglycan and LPS biosynthesis, respectively) were selectively up-regulated in the 48-h PI IFN-γ-treated samples. Taken together, these data supported the concept of a persistence stimulon in C. pneumoniae that is regulated at key points in various metabolic pathways. In addition to the analysis of biosynthetic genes, the up-regulated gene set from continuous C. pneumoniae TW-183 infections was also analysed in the validated IFN-γ-mediated C. pneumoniae A-03 persistence experiments. The data revealed similarities and differences in gene expression patterns between these two in vitro persistence models. Furthermore, the profiles obtained for genes such as pmp1 and porB provided insights into the widely predicted phenomenon of late developmental gene shut-down during chlamydial persistence. A final investigation into an analogous IFN-γ-mediated persistence system for C. trachomatis serovar L2 focussed on one up-regulated (murA) and one down-regulated (glmU) gene from the validated IFN-γ-mediated persistent C. pneumoniae A-03 data set. Both genes were significantly down-regulated in persistent C. trachomatis, adding to a growing body of evidence for key differences among chlamydial species in their persistent gene expression patterns. This project has contributed significantly to our understanding of the molecular basis of the important persistent phase of chlamydial development.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.

HMPV was first reported in Australia by Nissen et al in 2002 from a group of 200 nasopharyngeal aspirate (NPA) specimens collected throughout 2001 from children presenting to the Royal Children’s Hospital, Brisbane. These specimens, previously negative for all common viral pathogens, were screened for hMPV by a polymerase chain reaction (PCR) assay based on known sequences. Molecular diagnostic assays including conventional reverse transcriptase PCR assay (RT-PCR) and real-time RT-PCR assays were subsequently developed, and molecular characterisation studies in our laboratory identified four genetic groups of hMPV. At the start of this project, little information were available regarding the virological characteristics of hMPV such as the isolation and replication kinetics of the virus in eukaryotic cells, molecular assays capable of detecting all virus subtypes, quantitation of viral load, genotyping and molecular epidemiology, correlation between virus subtypes and disease severity, and clinical spectrum of the infection. This project was designed to elucidate the virological features of hMPV that had not been explained by earlier studies on this virus. The project was limited to retrospective studies utilising the sera and nucleic acids obtained from positive subjects presenting to our hospital. The project provided relevant data in these areas, which helped in the early detection of infection and treatment, and also provided information for future research on antibody profiles and vaccine development. The study examined specific areas related to clinical and virological characteristics of hMPV with the aim of applying the results in patient management. During the project, five areas of hMPV research were undertaken, addressing each through detailed studies. An outline of the project aims and the conclusions derived from those experimental chapters is described below: 1. Isolation of the virus from clinical specimens obtained from infected subjects An optimised tissue culture protocol was successfully developed for isolating hMPV from positive nasopharyngeal aspirates, using LLC-MK2 cell lines. Viral stocks were prepared and maintained at stable conditions for future experiments. The demonstration of virus infection in the eukaryotic cells and titration of the infectious virions were performed using immunological assays developed and optimised in our laboratory, during the course of this study. 2. The complete genome sequence of an Australian hMPV isolate In this study, we described the ‘13,333 base pair’ complete genome sequence of the Queensland hMPV type-A strain, designated as AUS-001. Phylogenetic analyses of individual genes were used to generate ‘topological trees’ for systematic comparison of our local hMPV strain to that of international sequences. 3. A quantitative PCR assay (q.PCR) for hMPV A quantitative real-time reverse transcription PCR assay (qrt.RT-PCR) was developed for the simultaneous detection and quantification of hMPV in clinical samples. Serial dilutions of a synthetic RNA control were amplified after determining the absolute RNA copy numbers, and a standard curve was derived based on the cycle thresholds (Ct) values of the respective dilutions. Quantification of the hMPV RNA in clinical specimens was performed by extrapolating this data with Ct values of specimen dilutions obtained from the real-time assay. The dynamic range of the assay for hMPV genotypes A and B was determined. Validation of the inter- and intra- assay variations was completed using negative and positive controls along with a second assay targeting a different gene. 4. Determine the molecular epidemiology of hMPV genotypes This component of the project was designed to determine the molecular epidemiology of Queensland hMPV strains, using a selected ‘specimen population of hMPV positives’ representing the period 2001 to 2004. An RT-PCR assay based on P gene regions of hMPV was developed for the molecular typing of the above panel. Analyses of nucleotide and predicted amino acid sequences confirmed the heterogeneity of hMPV strains. In our study group, two genotypes (A and B) further classified into four subtypes (A1, A2, B1 and B2), were found to co-circulate during this period. General epidemiological features of the hMPV infections including seasonality, co-infections, incidence and prevalence in different age groups and in general population were described. 5. Clinical characteristics of hMPV infections The aim of this analysis was to illustrate the clinical spectrum of hMPV infections in a Queensland study population. We described the hMPV incidence pattern in different age groups and investigated the clinical severity scores of hMPV genotypes based on reported clinical features. We also undertook to identify any correlations between disease severity and other factors, including genotype, co-infections and viral load. Summary On completion, this PhD study provided valuable data on the isolation, molecular detection, epidemiological pattern and clinical severity of hMPV infections in Queensland. Overall hMPV was determined to be a serious respiratory pathogen in Queensland children. Data from this thesis will contribute to improved patient management and reduce the burden of hMPV-related disease in Queensland. These studies also formed the basis of further research involving respiratory viral pathogens in our laboratory and nationally.

Με την ηλικία, νόσοι που σχετίζονται με δομικά ελαττώματα των οστών που οφείλονται σε κατάγματα ή εκφυλισμούς, αναμένονται να αυξηθούν σε συχνότητα. Επιπλέον, η αύξηση του προσδόκιμου ζωής επιβάλλει τη χρήση βελτιωμένων συνθετικών υλικών για την αντικατάσταση νοσούντων οστών, για παράδειγμα κατά τη χρήση μεταλλικών ράβδων σε περιπτώσεις βλαβών μη-ένωσης και στις χειρουργικές επεμβάσεις αντικατάστασης ισχίου. Τα υπάρχοντα υλικά σχετίζονται με υπο-βέλτιστη οστεοενσωμάτωση και προβληματική μακροπρόθεσμη επιβίωση του σύνθετου εμφυτεύματος. Για το λόγο αυτό, η βελτίωση των υλικών επικάλυψης και των μηχανικών ιδιοτήτων των νέων, κυτταρικά συμβατών, συστατικών είναι επιτακτική. Για να αντιμετωπιστεί αυτό το πρόβλημα, υλικά νέας γενιάς είναι διαθέσιμα, ενδεχομένως με καλύτερες ιδιότητες ως υπόστρωμα προσκόλλησης για τα κύτταρα των οστών. Ο σκοπός της παρούσας εργασίας ήταν να εκτιμηθεί η ικανότητα ενός νέου, ειδικά κατασκευασμένου υλικού απο νανοσωλήνες άνθρακα ως προς τη διατήρηση της σωστής έκφρασης των χαρακτηριστικών γονιδίων των οστεοβλαστών, με έμφαση στην έκφραση των γονιδίων που εμπλέκονται στις αλληλεπιδράσεις οστεοβλαστών-υποστρώματος και έτσι προωθούν την σταθερή προσκόλληση των κυττάρων στο υπόστρωμα. Παράλληλα, ερευνήσαμε και την επίδραση της μηχανικής καταπόνησης στην έκφραση των γονιδίων αυτών σε κύτταρα που καλλιεργήθηκαν σε νανοσωλήνες άνθρακα. Χρησιμοποιήσαμε δύο ανεξάρτητες απομονώσεις οστεοβλαστών διαφοροποιημένων από ανθρώπινα μεσεγχυματικά βλαστικά κύτταρα μυελού των οστών, δηλαδή προχωρήσαμε σε δύο ανεξάρτητα πειράματα. Και στα δύο, για να γίνει ο πειραματισμός όσο εγγύτερα στις πραγματικές συνθήκες, καλλιεργήσαμε τους οστεοβλάστες υπο στατικές συνθήκες όσο και υπό συνθήκες μηχανικής καταπόνησης, για την προσομοίωση "in vivo" συνθηκών, και συγκρίθηκε η γονιδιακή έκφραση οστεοβλαστών που καλλιεργήθηκαν σε πλαστικό έναντι επιφανειών επικαλυμμένων με νανοσωλήνες άνθρακα. Απομονώσαμε το RNA από τους οστεοβλάστες μετά από την καλλιέργειά τους για 3 και 24 ώρες και προσδιορίσαμε, χρησιμοποιώντας την τεχνική real time RΤ-PCR, την έκφραση των ακόλουθων γονιδίων σε επίπεδο mRNA: κολλαγόνο-α1, αλκαλική φωσφατάση, οστεοποντίνη, βινκουλίνη και ιντεγκρίνες α4, αV, β1 και β3. Συνολικά, τα αποτελέσματα της ανάλυσης του κυτταρικού mRNA έδειξαν ότι η γονιδιακή έκφραση μετά από 3 ώρες καλλιέργειας είναι πολύ μεταβλητή, και οριστικά συμπεράσματα δεν θα μπορούσαν να εξαχθούν. Ωστόσο, αφού δίνεται η ευκαιρία στα κύτταρα να προσκολληθούν σταθερά, στις 24 ώρες, κατέστη σαφές ότι: α) η κυτταρική ταυτότητα των διαφοροποιημένων οστεοβλαστών διατηρείται, με βάση το γεγονός ότι η έκφραση αυτών των χαρακτηριστικών γονιδίων, που σχετίζονται με την προσκόλληση, συντηρείται σωστά, αν και σε διάφορα επίπεδα, β) σε στατικές συνθήκες, το επίπεδο της έκφρασης των εξετασθέντων γονιδίων είναι κατά τι χαμηλότερο σε οστεοβλάστες που καλλιεργηθήκαν σε επικαλυμμένη επιφάνεια με νανοσωλήνες άνθρακα σε σύγκριση με τα κύτταρα που καλλιεργηθήκαν σε πλαστικό, και γ) σε σύγκριση με τις στατικές συνθήκες, το μηχανικό ερέθισμα ενισχύει την έκφραση αυτών των γονιδίων οστεοβλαστών όταν καλλιεργούνται σε νανοσωλήνες άνθρακα, για την επίτευξη υψηλών επιπέδων mRNA έκφρασης των γονιδίων κυτταρικής προσκόλλησης. Τα αποτελέσματα της τελευταίας ανάλυσης της γονιδιακής έκφρασης είναι επίσης συμβατά με τις συνολικές ποσότητες RNA που λαμβάνονται, υποστηρίζοντας έμμεσα τη σταθερή προσκόλληση και επιβίωση των οστεοβλαστών σε νανοσωλήνες άνθρακα υπο συνθήκες μηχανικής καταπόνησης. Συμπεραίνουμε λοιπόν ότι το νέο υπόστρωμα από νανοσωλήνες άνθρακα που αναλύθηκε σε μηχανικές συνθήκες διέγερσης που προσομοιάζουν, κατά το δυνατόν, συνθήκες καταπόνησης in vivo, συνιστά ένα κατάλληλο κυτταρικό υπόστρωμα, συμβατό με την επιβίωση των οστεοβλαστών, τη διαφοροποίηση, την ανάπτυξη και την σταθερή προσκόλλησή τους στο υπόστρωμα νανοσωλήνων. Η εργασία αυτή υποστηρίζει την πιθανότητα της χρήσης αυτών των νέων υλικών στο μέλλον για την επικάλυψη σκελετικών προσθέσεων, με σκοπό την απόκτηση βέλτιστης οστεοενσωμάτωσης. / As population ages, diseases related to bone structural defects due to fracture or degeneration are expected to increase in frequency. In addition, the increase in life expectancy necessitates better composite materials for replacement of diseased/fractured bones, for example during the use of metal rods for non-union defects and in hip replacement surgery. The existing materials are associated with sub-optimal osseointegration and problematic long-term survival of the composite graft. For this reason, improvement of coating materials and engineering of novel cell-compatible components is imperative. To address this problem, new-generation materials are available, with possibly better bone cell adherence properties. The Aim of this work was to evaluate the ability of a novel, specially-constructed carbon nanotube material to sustain proper expression of characteristic osteoblast genes, with emphasis on the expression of genes that are functionally involved in osteoblast-matrix interactions and promote firm cell adherence to substrate. We used two independent isolates of osteoblasts differentiated from human bone marrow mesenchymal stem cells, ie we proceeded to two independent experimental runs. In both, to make the experimentation more context-relevant, we grew the osteoblasts in static as well as under mechanical strain, to simulate in vivo conditions, and also compared gene expression in osteoblasts grown on plastic versus carbon nanotube-coated surface. We isolated RNA from the osteoblasts at 3 hours and 24 hours after seeding them on the culture vessels and determined, using real-time RT-PCR techniques, the level of expression of the following genes at the mRNA level: α1-collagen, alkaline phosphatase, osteopontin, vinculin, and integrins α4, αV, β1 and β3. All in all, the results on cell mRNA analysis indicated that gene expression at 3h post-plating is too variable and no firm conclusions could be drawn. However, once the cells are given a chance to firmly adhere, at 24h, it became clear that: a) osteoblast cell identity is maintained, based on the fact that the expression of these characteristic matrix- and adhesion-related genes is properly maintained, albeit in various levels, b) in static conditions, the level of expression of the examined genes is lower in cells grown on nanotube-coated surface compared to cells grown on plastic, and c) in comparison to static conditions, mechanical stimulation enhances expression of these genes in osteoblasts grown on nanotubes, to attain robust levels of cell adherence gene mRNA expression. The results of the latter gene expression analysis are also compatible with total RNA quantities obtained, indirectly arguing firm osteoblast adhesion/survival on nanotubes under mechanical strain conditions. We therefore conclude that the novel carbon nanotubes assayed herein in lifelike mechanical stimulation conditions, constitute an appropriate cell-bearing surface, compatible with osteoblast survival, differentiation, growth and firm adherence to substrate. This work raises the possibility of using this novel material in the future to coat skeletal prostheses, in order to obtain improved osseointegration.

Fundação de Amparo a Pesquisa do Estado de São Paulo / The reverse transcription polymerase chain reaction (RT-PCR) of turkey astrovirus (TAstV) capsid and polymerase gene was applied in the bursa of Fabricius (BF), thymus (TH), spleen (SP) and cloacal swabs (CS) from young poults with PEMS (Poult Enteritis Mortality Syndrome). The histological lesions were atrophy, lymphoid depletion, cellular infiltration and necrosis of BF, TH and SP, respectively. The RT-PCR reactions were positive in all of 100 CS, 7 out of 10 of BF, 10 out of 20 TH and SP, respectively, for the polymerase gene of TAstV-2. Five out of 10 TH and SP samples, considered as negative by RT-PCR, were positive when specific primers designed to the TAstV-2 capsid gene were applied. Finally, this is the first description of turkey astrovirus infection presenting PEMS in Latin America. / O Astrovírus dos Perus Tipo 2 (TAstV-2) é agente etiológico de uma doença emergente em perus, a Poult Enteritis Mortality Syndrome (PEMS), caracterizada por enterite severa, elevados índices de mortalidade, atrofia linfóide e imunossupressão em aves jovens, sendo responsável por sérios prejuízos financeiros à indústria avícola dos Estados Unidos. No Brasil, há necessidade de conhecimento sobre a ocorrência desses vírus nas diarréias das aves, sendo esse, um passo fundamental para o estabelecimento de medidas profiláticas específicas e para a exportação de produtos avícolas dentro das condições de sanidade. Este estudo teve por objetivos detectar TAstV, pela técnica de RT-PCR dirigida aos genes codificadores da polimerase e capsídeo viral, em perus jovens de 30 a 45 dias de idade com quadro clínico de diarréia severa, imunossupressão, baixo desempenho zootécnico e mortalidade, caracterizar alterações histopatológicas em bursa de Fabrícius (BF), timo (T) e baço (B) e, por fim, comparar os resultados obtidos com os dados de literatura. O TAstV-2 foi detectado em todas amostras de swabs cloacais (SC, n=100), 7 amostras de bursa de Fabrícius (BF, n=10) e em 10 amostras de timo (T, n=20) e baço (B, n=20). De 10 amostras de timo e baço negativas na primeira análise de RT-PCR, 5 foram positivas com o uso de primers específicos para o gene do capsídeo do TAstV-2. Os exames histológicos revelaram ocorrência de atrofia, depleção linfóide, infiltração celular e necrose da BF, T e B, respectivamente. Esses resultados representam a primeira descrição do TAstV-2 circulante em lotes comerciais de perus na América Latina. / Mestre em Genética e Bioquímica

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-01-31Bitstream added on 2014-06-13T20:58:38Z : No. of bitstreams: 1 sanches_mm_me_botfca.pdf: 690574 bytes, checksum: 4794d7c23f85fb7d5e54fb4b30c47365 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Empresa Privada / Recentemente sintomas da doença conhecida como engrossamento das nervuras, ou big-vein, foram observados no Estado de São Paulo, principalmente no período de inverno. A doença foi historicamente associada ao Lettuce big-vein associated virus (LBVaV), porém a presença dos sintomas característicos foi atribuída ao Mirafiori lettuce big-vein virus (MLBVV). Tradicionamente ambos vírus vem sendo diagnosticados pelo teste de ELISA, de modo que resultados discrepantes quanto ao agente causal dos sintomas da doença foram obtidos na Europa, possivelmente devido à falta de sensibilidade do teste. Deste modo, o presente trabalho teve como finalidade utilizar a técnica de RT-PCR na detecção segura e específica do MLBVV e LBVaV. Foram coletadas 366 plantas sintomáticas nas regiões produtoras de Bauru, Campinas e Mogi das Cruzes no Estado de São Paulo nos meses de junho e setembro de 2004 e abril e julho de 2005, e 18 plantas assintomáticas na região de Mogi das Cruzes no mês de dezembro de 2004. Os oligonucleotídeos específicos foram altamente eficientes na detecção de ambos os vírus, sendo que a banda viral foi clonada e sequenciada para alguns dos isolados, comprovando a identidade viral de cada um dos vírus. Foi observado que 76,2% das plantas sintomáticas apresentaram infecção mista do LBVaV e MLBVV, em 11,5% somente o MLBVV e em 6,6% somente o LBVaV. Nas plantas assintomáticas foi detectada a presença de infecção mista por MLBVV e LBVaV em quatro amostras, infecção apenas por MLBVV em cinco amostras e apenas por LBVaV em três amostras, indicando que o desenvolvimento de sintomas depende de fatores abióticos, além da presenca dos vírus. A análise das sequencias de aminoácidos da região codificadora da proteína capsidial, revelou que os isolados de LBVaV possuem baixa variabilidade genética e mesma origem evolutiva entre isolados de diferentes partes do mundo... / Lettuce plants with big-vein symptons have been observed in the São Paulo State during the winter. The disease has been historically associated to Lettuce big-vein associated virus (LBVaV), however recently the development of symptoms was atributted to Mirafiori lettuce big-vein virus (MLBVV). Tradicionally both viruses were routinely detected by ELISA, but discrepants results about the main disease agent were obtained in the Europe possible by the low sensibility of the assay. The objective of this study was to detect MLBVV and LBVaV by RT-PCR, using specifics primers. A total of 366 samples from symptomatic plants of Bauru, Campinas and Mogi das Cruzes regions, from São Paulo State, were collected during june and september of 2004 and april and july of 2005, and 18 symptomless plants from the Mogi das Cruzes region during December 2004. The primers were highly efficient in the 4 detection of both viruses, and the fragment of some isolates were cloned and sequenced to confirm the RT-PCR. Mixed infection of LBVaV and MLBVV was observed on 76,2% symptomatic plants. MLBVV on 11,5% and LBVaV on 6,6%. In a total of 18 symptomless plants, four were infected with both viruses, five only with MLBVV and three plants with LBVaV. These results indicates that not only the presence of the viruses, but also abiotic factors are necessary for the occurrence of big-vein symptoms. Amino acid sequence identities of part of the coat protein gene of LBVaV isolates was high indicating a possible same evolutive origin. Genetic diversity among MLBVV isolates was higher when compared to LBVaV isolates. MLBVV brazilian isolates belongs to subgroup A, with one RsaI restriction site on the coat protein sequence. One plant with MLBVV and LBVaV (mixed infection) was sap inoculated on a host range (temperature and luminosity controled), indicating that MLBVV can be transmitted to Nicotiana tabacum TNN, N. rustica... (Complete abstract, click electronic address below).

Orientador: Renate Krause Satake / Banca: Marcelo Agenor Pavan / Banca: Romulo Fujito Kobori / Resumo: Recentemente sintomas da doença conhecida como engrossamento das nervuras, ou big-vein, foram observados no Estado de São Paulo, principalmente no período de inverno. A doença foi historicamente associada ao Lettuce big-vein associated virus (LBVaV), porém a presença dos sintomas característicos foi atribuída ao Mirafiori lettuce big-vein virus (MLBVV). Tradicionamente ambos vírus vem sendo diagnosticados pelo teste de ELISA, de modo que resultados discrepantes quanto ao agente causal dos sintomas da doença foram obtidos na Europa, possivelmente devido à falta de sensibilidade do teste. Deste modo, o presente trabalho teve como finalidade utilizar a técnica de RT-PCR na detecção segura e específica do MLBVV e LBVaV. Foram coletadas 366 plantas sintomáticas nas regiões produtoras de Bauru, Campinas e Mogi das Cruzes no Estado de São Paulo nos meses de junho e setembro de 2004 e abril e julho de 2005, e 18 plantas assintomáticas na região de Mogi das Cruzes no mês de dezembro de 2004. Os oligonucleotídeos específicos foram altamente eficientes na detecção de ambos os vírus, sendo que a banda viral foi clonada e sequenciada para alguns dos isolados, comprovando a identidade viral de cada um dos vírus. Foi observado que 76,2% das plantas sintomáticas apresentaram infecção mista do LBVaV e MLBVV, em 11,5% somente o MLBVV e em 6,6% somente o LBVaV. Nas plantas assintomáticas foi detectada a presença de infecção mista por MLBVV e LBVaV em quatro amostras, infecção apenas por MLBVV em cinco amostras e apenas por LBVaV em três amostras, indicando que o desenvolvimento de sintomas depende de fatores abióticos, além da presenca dos vírus. A análise das sequencias de aminoácidos da região codificadora da proteína capsidial, revelou que os isolados de LBVaV possuem baixa variabilidade genética e mesma origem evolutiva entre isolados de diferentes partes do mundo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Lettuce plants with big-vein symptons have been observed in the São Paulo State during the winter. The disease has been historically associated to Lettuce big-vein associated virus (LBVaV), however recently the development of symptoms was atributted to Mirafiori lettuce big-vein virus (MLBVV). Tradicionally both viruses were routinely detected by ELISA, but discrepants results about the main disease agent were obtained in the Europe possible by the low sensibility of the assay. The objective of this study was to detect MLBVV and LBVaV by RT-PCR, using specifics primers. A total of 366 samples from symptomatic plants of Bauru, Campinas and Mogi das Cruzes regions, from São Paulo State, were collected during june and september of 2004 and april and july of 2005, and 18 symptomless plants from the Mogi das Cruzes region during December 2004. The primers were highly efficient in the 4 detection of both viruses, and the fragment of some isolates were cloned and sequenced to confirm the RT-PCR. Mixed infection of LBVaV and MLBVV was observed on 76,2% symptomatic plants. MLBVV on 11,5% and LBVaV on 6,6%. In a total of 18 symptomless plants, four were infected with both viruses, five only with MLBVV and three plants with LBVaV. These results indicates that not only the presence of the viruses, but also abiotic factors are necessary for the occurrence of big-vein symptoms. Amino acid sequence identities of part of the coat protein gene of LBVaV isolates was high indicating a possible same evolutive origin. Genetic diversity among MLBVV isolates was higher when compared to LBVaV isolates. MLBVV brazilian isolates belongs to subgroup A, with one RsaI restriction site on the coat protein sequence. One plant with MLBVV and LBVaV (mixed infection) was sap inoculated on a host range (temperature and luminosity controled), indicating that MLBVV can be transmitted to Nicotiana tabacum TNN, N. rustica... (Complete abstract, click electronic address below). / Mestre

In Brazil there is still no variety of commercial transgenic sugarcane used in large scale, although biotechnological research in the area of gene expression has been performed in order to obtain varieties that can be cultivated in low rainfall, high temperatures and in low fertil soils. In the present work it was analised the diferencial gene expression in leaves of the variety of sugarcane RB72910 in field capacity and under severe water stress in a greenhouse. The evaluation was done using the technique real-time RT-PCR a powerful tool used to identify and quantify significant changes in the levels of transcripts, facilitating the selection of candidate genes for use in transgenic plants. For this we analized the expression of 11 genes in leaf samples in two conditions of water regime. The genes analyzed were: DNAJ, PGR5, H1, PSI, LTP, WIP, ZmPIP2-1, ZmTIP4-2, SAMDC and two genes with unknown functions. It was observed that the variety studied showed diferential gene expression under water stress mainly for genes encoding proteins of the protective fotosynthetic system and for maintenance of the homeostasis. Thus, it was concluded that the genotype RB72910 has important agronomical traits of fotoprotection and adaptation to drought. / Fundação de Amparo a Pesquisa do Estado de Alagoas / No Brasil ainda não há uma variedade de cana-de-açúcar transgênica comercial usada em larga escala, porém pesquisas biotecnológicas na área de expressão gênica estão sendo feitas visando a obtenção de variedades que possam ser cultivadas em condições de baixa pluviosidade, altas temperaturas e solos com pouca fertilidade. No presente trabalho foi analisada a expressão gênica diferencial em folhas na variedade de cana-deaçúcar RB72910 nas condições de capacidade de campo e sob estresse hídrico severo em casa de vegetação. A avaliação foi feita por meio da técnica real-time RT-PCR, uma potente ferramenta usada para identificar e quantificar mudanças significativas nos níveis de transcritos, facilitando a seleção de genes candidatos à utilização em transgenia. Para isso foi analisada a expressão de 11 genes em amostras foliares em duas condições de regime hídrico. Os genes analisados foram: DNAJ, PGR5, H1, PSI, LTP, WIP, ZmPIP2-1, ZmTIP4-2, SAMDC e dois genes com funções indeterminadas. Observou-se que a variedade estudada demonstrou expressão diferencial sob estresse hídrico principalmente para genes que codificam proteínas de proteção do sistema fotossintético e de manutenção da homeostase. Com isso, concluímos que o genótipo RB72910 apresenta importantes características agronômicas de fotoproteção e de adaptação à seca.