The ability of bacteria to assimilate free DNA from the environment is known as competence. Though many studies have focused on competence regulation in Streptococcus pneumoniae and Streptococcus gordonii, Streptococcus sanguinis has yet to be examined. Physiological characterization of competence in S. sanguinis strain SK36 and its comC mutant, JFP41, led to the genome-wide transcriptional analysis of cells induced to competence via addition of competence-stimulating peptide (CSP). A total of 128 genes were induced at least 2-fold, 74 of which were classified as either “early” or “late” based on their induction patterns. Expression patterns were verified using qRT-PCR. This study identified genes not up-regulated in S. pneumoniae or S. gordonii and lays the foundation for bioinformatic studies to identify conserved binding sites upstream from CSP-regulated genes. These results also shed light on the possible existence and identity of expected CSP exporters in S. sanguinis, which have so far eluded discovery.

Η σύγκριση του μεταγραφικού προφίλ με χρήση μικροσυστοιχιών DNA είναι μια τεχνική που βελτιστοποιήθηκε την τελευταία δεκαετία. Η διαδικασία μπορεί να διαχωριστεί σε δύο μέρη: το πειραματικό και το υπολογιστικό. Το πρώτο μέρος αφορά στα βήματα της επιλογής της μικροσυστοιχίας, της απομόνωσης του βιολογικού υλικού, της σήμανσης και της επέκτασης, της υβριδοποίησης και της σάρωσης. Το υπολογιστικό μέρος αφορά στην ποσοτικοποίηση της εικόνας, στην κανονικοποίηση των δεδομένων και στην ανάλυσή τους. Πριν από τα παραπάνω βήματα πρέπει να έχει διατυπωθεί το κατάλληλο βιολογικό ερώτημα. Το βιολογικό ερώτημα που τέθηκε αφορούσε στις διαφορές στη γονιδιακή έκφραση των γενετικά τροποποιημένων εμβρυικών ινοβλαστών του ποντικού οι οποίοι έφεραν έλλειψη του ενός αλληλομόρφου της πρωτεΐνης Geminin σε σύγκριση με ομόζυγους εμβρυικούς ινοβλάστες. Για το σκοπό αυτό επιλέχθηκε μικροσυστοιχία δοκιμής με 384 ανιχνευτές γονιδίων ποντικού σε διπλά αντίγραφα. Απομονώθηκε ολικό RNA από τα κύτταρα, σημάνθηκε με απ’ ευθείας σήμανση και οι cDNA στόχοι που προέκυψαν, υβριδοποιήθηκαν στη μικροσυστοιχία. Έπειτα από δοκιμές καθορίστηκαν οι κατάλληλες συνθήκες για ένα ολοκληρωμένο πείραμα σύγκρισης μεταγραφικού προφίλ. Στη συνέχεια, πραγματοποιήθηκε προκαταρκτική ανάλυση του πειράματος η οποία περιελάμβανε ποσοτικοποίηση της εικόνας που προέκυψε, κανονικοποίηση των δεδομένων και πολυπαραμετρική ανάλυσή τους. / Transcriptional profiling using DNA microarrays was optimized during the last decade. The process can be separated in two parts: the experimental part and the computational part. The first part includes the steps of microarray choice, isolation of the biological material, labeling and amplification, hybridization and scanning. The computational part includes image quantification, data normalization and data analysis. Before the above steps, an appropriate biological question should be formulated. We wished to study differences in the gene expression profile of genetically modified mouse embryonic fibroblasts bearing a knock – out allele of the Geminin gene (homozygote|+/- Gem), in comparison to wild type embryonic fibroblasts. For this reason, a trial microarray was chosen with 384 gene probes in duplicates. Total RNA was isolated from the cells, directly labeled and the cDNA targets synthesized were hybridized to the microarray. After a series of trials, optimal conditions for a complete transcriptional profiling experiment were determined. Initial analysis including quantification of the image, data normalization and further multifunctional analysis was then carried out.

Orientador: Sergio Roberto Peres Line / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-13T02:01:37Z (GMT). No. of bitstreams: 1 Almeida_CarolinaVieirade_M.pdf: 673929 bytes, checksum: de7a7d8f324324fae24215bb7eb7ed66 (MD5) Previous issue date: 2009 / Resumo: O gene PAX9 pertence a uma família de fatores de transcrição denominada família Pax. Esse gene é expresso em tecidos embrionários como somitos, bolsa endodérmica da faringe, brotos distais dos membros e mesênquima derivado da crista neural. Alguns polimorfismos na região promotora desse gene em humanos vêm sendo associados a formas de agenesias não-sindrômicas autossômicas dominantes. No presente trabalho, nós verificamos a expressão gênica e a influência de duas seqüências da região promotora na transcrição do gene PAX9 em ensaios in vitro com células de tecido embrionário de ratos de 13.5 dias obtidas a partir de seus membros, face e regiões do mesencéfalo e romboencéfalo. Os fragmentos da região promotora foram clonados em plasmídios repórteres e transfectados nos três diferentes tecidos embrionários. Nossos resultados das análises de RT-PCR demonstraram que em culturas in vitro, as células dos membros e do SNC expressam o gene PAX9, porém, aquelas obtidas a partir da face não o expressaram. Além disso, os resultados dos ensaios de luciferase mostraram que a atividade dessa nos vetores construídos são mais fracas do que do vetor pGL3-Basic sozinho, sugerindo que essas regiões não são suficientes para guiar a transcrição gênica. / Abstract: PAX9 belongs to a transcriptional factor genes family named Pax. This gene is expressed in embryonic tissues like somites, pharyngeal pouch endoderm, distal limb buds and neural crest-derived mesenchyme. Some polymorphisms in the upstream promoter region of the human PAX9 have been associated with human non-syndromic forms of autossomal dominant agenesis. In the present study we verified the in vitro expression of this gene and the influence of two promoter sequences in the transcription of PAX9 gene, in embryo tissues obtained from digits, face and midbrain and hindbrain regions. These fragments were cloned on reporter plasmid and were transfected into the different cells cultures. Our RT-PCR results showed that in vitro E13.5 limb bud and CNS cells express PAX9, but not in derived facial region cells. Moreover, the luciferase activity of the Pax9 promoter vectors were weaker than pGL3-Basic alone, suggesting that the PAX9 sequences of promoter region are not sufficient to drive PAX9 gene transcription. / Mestrado / Histologia e Embriologia / Mestre em Biologia Buco-Dental

Maintenance of the levels of nucleoside triphosphates (NTPs) as well as their corresponding deoxy derivatives (dNTPs) is crucial to all growth and developmental processes. The enzyme nucleoside diphosphate kinase (NDK) utilises an autophosporylated enzyme intermediate to catalyse the transfer of 5’ terminal phosphate from NTPs (mostly ATP) to nucleoside diphosphates (NDPs) via a reversible mechanism as given below. N1TP + NDK ↔N1DP+ −NDK-His* (1) N2DP + NDK-His* P ↔N2TP + NDK−His. (2) In the γ-phosphoryl group transfer, the highly conserved His 117 active site residue becomes autocatalytically phosphorylated, in the enzyme intermediate (NDK-H*). This phosphoryl group is transferred to ribo-or deoxyribonucleotides (N2DP) in a substrate non-specific manner. In addition to its fundamental role in nucleotide metabolism, NDP kinase is also involved in a number of cellular regulatory functions such as growth and developmental control, tumor metastasis suppression, signal transduction and so on. From mycobacterial genera, NDK of Mycobacterium tuberculosis (MtNDK) has been crystallised, structure was solved and biochemical functions were elucidated. However, there has not been any such study on the NDK of Mycobacterium smegmatis, except on the possible interaction with other proteins which modulates the NTP synthesising activity of MsNDK, towards specific NTPs. M. smegmatis, being a saprophytic, fast growing and non-pathogenic mycobacterium that is widely used as an experimental model mycobacterial system to study various biological processes in mycobacteria, it was thought appropriate to study NDK from this organism. The outcome of current study is presented in five chapters. The First Chapter gives a detailed introduction on the structural and functional aspects of NDK from diverse organisms, from bacteria to humans. Chapter 2. Molecular Cloning, Expression and Characterisation of Biochemical Activities of Nucleoside Diphosphate Kinase from Mycobacterium smegmatis mc 155 The research work starts with the molecular cloning, overexpression, purification, and characterisation of biochemical activities of recombinant MsNDK protein. In brief, ndk gene from M. smegmatis (Msndk) has been cloned, efficiently overexpressed as a soluble 6xHis-tagged recombinant protein, purified through affinity chromatography, and its biochemical characterisation for ATPase, GTPase and NTP synthesising activities have been demonstrated. Catalytic mutant of MsNDK, MsNDK-H117Q, was generated using site-directed mutagenesis approach and H117 was shown to be essential for the catalytic activity. Further experiments revealed that it is the same H117 that is required for mediating autophosphorylation as well, which is an intermediate in the transphosphorylation reaction of NDK. Chapter 3. Characterisation of Oligomerisation Property of M. smegmatis Nucleoside Diphosphate Kinase: the Possible Role of Hydrogen Bond and Hydrophobic Interactions The present study revealed that presence of homodimer of MsNDK could be observed in the presence of heat and SDS. Chemical cross-linking experiments revealed that MsNDK forms dimer, tetramer and hexamer. Homology modeling of MsNDK on the MtNDK crystal structure supported the existence of hexamer as three homodimers. Gln 17, Ser 24 and Glu 27 were found to be positioned at the dimer interface. Mutations on these residues did not abolish the stability of the respective mutant dimers in the presence of SDS and heat. Modeled structure of MsNDK revealed the existence of hydrophobic interactions at the dimer interface. In silico approach helped in mapping the existence of hydrophobic interactions at the dimer interface as two consecutive β-strands. Exposure of hydrophobic residues, using organic solvent methanol, abolished the dimer completely, indicating the vital role of hydrophobic interactions in the dimer stability. In solution, the native MsNDK was found to be a hexamer. Chapter 4. Mycobacterial Nucleoside Diphosphate Kinase Functions as GTPase Activating Protein for Mycobacterial Cytokinetic Protein FtsZ In Vitro Mammalian, plant, and bacterial NDKs can function as GTPase activating protein (GAP) for small G proteins namely, p21 Ras, Rad, and Rho-GTPases in animals and Pra1, Pra2, and GPA1 in Arabidopsis thaliana in vitro. We examined whether NDK of M. tuberculosis (MtNDK) can function as GAP in vitro for the cytokinetic protein FtsZ of Mycobacterium tuberculosis (MtFtsZ), which is a protein with a classical G-protein fold, possessing GTP-binding and GTPase activities (like G proteins). Both MtNDK and MsNDK could function as GAP for MtFtsZ and FtsZ of M. smegmatis (MsFtsZ) respectively in vitro. Similarly, MtNDK could function as GAP for MsFtsZ and reciprocally MsNDK could function as GAP from MtFtsZ. Interaction of NDK with respective FtsZ could be observed. Physiological implications of GAP activity of NDK on FtsZ are discussed. Chapter 5. Transcriptional Analyses of Nucleoside Diphosphate Kinase Gene of Mycobacterium smegmatis mc 155 Although there are studies on the structural and functional aspects of NDK, there are not many studies available on the transcriptional analysis of nucleoside diphosphate kinase (NDK) gene expression in general and nothing in particular in mycobacterial systems. Therefore we studied the transcriptional analysis of expression of Msndk gene, in order to map the Transcriptional Start Site (TSS), identification of promoter elements, and elucidated of transcriptional activity of the promoters. Expression of Msndk gene was analysed in exponential growth phase and under two different stress conditions wherein DNA replication gets arrested. Hydroxy Urea (HU), which reduce dNTP pools by inhibiting ribonucleotide reductase and Phenethyl Alcohol (PEA), which affects membrane structure resulting in DNA replication arrest, were used. Two transcripts and their promoter elements were mapped and their promoter activities were demonstrated. The profile of transcripts was found to be identical under the three different conditions examined.

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