The parathyroid hormone related protein (PTHrP) and Indian Hedgehog (IHH) are two secreted molecules, acting as paracrine factors during embryonic development and post-natal growth of endochondral bones. PTHrP and IHH are essential factors for the regulation of chondrocyte proliferation and differentiation. However, it has previously been shown that PTHrP and IHH are also expressed in the chick and mouse embryos intramembranous bones, which do not form through a cartilage intermediate and in which chondrocytes are absent. Similarly, the zebrafish orthologs, pthrp1 and ihha, are also expressed during the regeneration of the intramembranous bones of the fin rays of the zebrafish caudal fin. This surprising observation led us to further analyze the expression and function of pthrp1 and ihha in the regenerating fin rays. Gene expression analysis using in situ hybridization shows that pthrp1 is expressed in a stripe of cells located within the domain of expression of ihha in the newly differentiating osteoblasts in the regenerating fin rays. Also, pthrp1 expression is observed at the level of the joints between the bone segments forming the rays and co-localizes with the expression domain of evx1, a transcription factor that has been implicated in the formation of joints in the caudal fin. Furthermore, RT-PCR analyses show that pthrp2 and the pthrp receptors mRNA (pth1r, pth2r and pth3r) are also present in the fin regenerate. Finally, functional analysis shows that the knockdown of pthrp1 or ihha expression by electroporation of morpholinos induces a delay of the regenerative outgrowth of the fin. These results suggest that pthrp1 and ihha may be involved in the regulation of proliferation and differentiation of chondrocyte-like osteoblasts in the fin rays, playing a role similar to that described in the mammalian growth plate of endochondral bones. In addition, pthrp1 is possibly an important factor involved in the formation and maintenance of joints of the dermal bones of the fin rays.

Les nématodes à kystes sont l'un des bioagresseurs causant le plus de dégâts sur les cultures de pommes de terre. La résistance trouvée chez l'accession spl88S.329.18, issue de l'espèce Solanum sparsipilum est caractérisée par un déterminisme oligogénique avec un QTL à localisé sur le chromosome V (GpaVspl) et un QTL mineur localisé sur le chromosome XI(GpaXIspl). Pour obtenir une résistance de haut niveau, l'effet du QTL GpaVspl, doit êtrecomplémenté par celui du QTL à effet faible GpaXIspl. Par génomique comparative, le locusGpaV a été localisé dans un intervalle compris entre 16 et 60 kb sur les génomes de la tomateet des espèces apparentées à la pomme de terre, Solanum demissum et Solanum phureja. Deuxgènes ont été annotés dans cet intervalle sur les génomes de la tomate et de S. demissum : lepremier appartient à la famille des TIR-NBS-LRR (TNL), famille de gènes de résistanceclassiques, et le second appartient à la famille des " mitochondrial, transcription terminationfactor " (mTERF), dont l'implication dans des mécanismes de résistance n'a jamais étédémontrée.Les objectifs de ma thèse étaient d'identifier le(s) gène(s) responsable(s) de la résistance àG. pallida, conférée par le locus GpaVspl, et d'étudier sa régulation. Suite à la publication de laséquence du génome de S. phureja, en 2011, nous avons mis en évidence que le locus GpaVétait dupliqué chez S. phureja et que cette duplication était également présente chezS. sparsipilum. Les quatre gènes annotés au locus GpaVspl ont été nommésSpl_mTERF18430, Spl_TNL18429, Spl_mTERF18453 et Spl_TNL18428.L'effet des deux gènes Spl_mTERF18430 et Spl_TNL18428 sur la résistance à G. pallida aété analysé via des expériences de transformation génétique suivies par des tests de résistancesur les plantes transformées. Un effet partiel du gène Spl_TNL18428 sur la résistance àG. pallida a été mis en évidence par complémentation de plantes sensibles. Aucun effetsignificatif n'a été détecté pour le gène Spl_mTERF18430. Des expériences d'extinctiongénique suggèrent que le deuxième gène TIR-NBS-LRR, Spl_TNL18429, qui est égalementexprimé dans les racines et qui présente un fort pourcentage d'identité de séquence avec legène Spl_TNL18428, pourrait également être impliqué dans la résistance à G. pallida.L'expression du gène rapporteur GFP, placé sous le contrôle du promoteur du gèneSpl_TNL18428, est fortement induite dans les cellules situées autour du syncytium. Cecirenforce l'hypothèse d'une implication du gène Spl_TNL18428 dans la résistance à G. pallida,car la localisation de l'expression de la GFP est similaire à celle de la nécrose, qui estcaractéristique de la réaction développée par les plantes résistantes autour du syncytium induitpar les nématodes.En tenant compte des données bibliographiques récentes, montrant que plusieurs gènes NBSLRRpeuvent être indispensables à l'expression d'une résistance, nos résultats suggèrent queles deux gènes Spl_TNL18428 et Spl_TNL18429 sont nécessaires à l'expression de larésistance à G. pallida

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.

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