No description available.

In nature, biofilm is a complex structure resulted of multicellular bacterial communities that provide important nutritional functions and the acquisition of protective traits such as antibiotics resistance and horizontal gene transfer. The development from the planktonic, lonely bacteria, to the mature multilayered biofilm structure consists of three main phases: motility, attachment and biofilm maturation. At cellular level, the process is controlled by several genes such as flhD, fliA, rpoS, csgD, adrA, cpxR all acting as master regulators. Additionally, the global regulators CRP, IHF, Fis, and others in less frequency, have been related to biofilm formation, although blurry information has been provided. In this thesis we used synthetic, molecular and cellular biology approaches to understand the effect of CRP, IHF and Fis in the transcriptional regulatory network in the bacterium Escherichia coli. In the first chapter, we employed network analysis to reconstruct and analyze part of the entire regulatory network described to modulate the flagella-biofilm program. With this analysis we identified some critical interactions responsible for the planktonic-biofilm transition. Next, we selected the top ten effectors nodes of the network and cloned the promoter region of those genes in a reporter system. As extensively explained in chapter II, this system allowed us to validate as well as suggest new interactions in the network. Additionally, the measurement of the promoter activity during bacterial development show that CRP, IHF and Fis differentially modulate most of the surveyed genes suggesting that those Global Regulators participate to modulate gene expression in different phases of the planktonic-biofilm development. At chapter three, to get a better overview of the entire process, we performed motility, adherence/early biofilm and mature biofilm assays. We describe the intrinsic ability of E. coli to perform motility, adherence and mature biofilm at 37?C. In contrast, the absence of ihf, fis as well as Carbon Catabolite Repression (CCR), lead to altered phenotypes at both motility and biofilm development. At the end, we discussed how the changes of promoter activity of target genes, together with our network analysis, could explain part of the altered phenotypes observed. For instance, we observed changes at the main stress responders rpoS and rpoE that, in combination with alterations at specific genes such as fliA, can explain the enhanced motility in the E. coli ?ihf strain. Altogether, in this thesis, we provided evidence that CRP, IHF and Fis control the activity of the promoter regions of genes involved in the planktonic-biofilm development. / Na natureza, o biofilme é uma estrutura complexa resultante de comunidades bacterianas multicelulares que fornece importantes funções nutricionais e a aquisição de traços de proteção como resistência a antibióticos e transferência horizontal de genes. O desenvolvimento das bactérias planctônicas solitárias para uma estrutura de biofilme maduro consiste em três fases principais: motilidade, fixação e maturação do biofilme. Ao nível celular, o processo é controlado por vários genes tais como flhD, fliA, rpoS, csgD, adrA, cpxR, todos agindo como reguladores mestre. Além disso, os reguladores globais CRP, IHF, Fis e outros em menor freqüência, têm sido relacionados à formação de biofilme, embora tenham sido fornecidas informações nao conclusivas sobre esse processo. Nesta tese foram utilizadas abordagens de bioinformática, assim como de biologia molecular e celular para entender o efeito de CRP, IHF e Fis na rede reguladora da transição de motilidade para biofilme na bactéria Escherichia coli. No primeiro capítulo, utilizamos a análise de rede para reconstruir e analisar parte da rede regulatória descrita para modular o programa flagelo-biofilme. Com esta análise identificamos algumas interações críticas responsáveis pela transição planctônica-biofilme. Em seguida, selecionamos os dez principais nós efetores da rede e clonamos a região promotora desses genes em um sistema repórter. Conforme explicado amplamente no capítulo II, este sistema nos permitiu validar e sugerir novas interações na rede. Adicionalmente, a medição da atividade do promotor durante o desenvolvimento bacteriano mostra que a CRP, a IHF e a Fis modulam diferencialmente a maioria dos genes analisados sugerindo que estes Reguladores Globais participam para modular a expressão génica em diferentes fases do desenvolvimento de estado planctónico para biofilme. No capítulo três, para obter uma melhor visão geral de todo o processo, realizamos ensaios de motilidade, aderência / biofilme precoce e biofilmes maduros. Descrevemos a capacidade intrínseca de E. coli para realizar motilidade, adesão e biofilme maduro a 37 °C. Em contraste, a ausência de ihf, fis, bem como o fenômeno de Repressão de Catabolite de Carbono (CCR), levam a fenótipos alterados, tanto na motilidade como no desenvolvimento do biofilme. No final, discutimos como as mudanças da atividade do promotor de genes alvo, juntamente com a nossa análise de rede, poderia !xi explicar parte dos fenótipos alterados observados. Por exemplo, observamos mudanças nos principais respondedores de estresse rpoS e rpoE que, em combinação com alterações em genes específicos como fliA, podem explicar a motilidade aumentada na estirpe de E. coli ?ihf. Em conjunto, nesta tese, apresentamos evidências de que CRP, IHF e Fis controlam a atividade das regiões promotoras de genes envolvidos no desenvolvimento planctônico-biofilme.

Mycoplasma hyopneumoniae é uma bactéria de tamanho diminuto, caracterizada por um genoma pequeno, com baixo conteúdo GC. Está associada com doenças respiratórias de suínos, resultando em prejuízos produtivos e econômicos na indústria animal. A presença de sequências de DNA repetitivas, que ocorrem em grandes quantidades em células eucarióticas, vem sendo cada vez mais identificadas em genomas de procariotos, sendo também associadas a um potencial papel regulador. Uma vez que a regulação da transcrição nesses organismos ainda é pouco entendida, o objetivo do presente estudo foi realizar uma busca in silico por elementos repetitivos nas regiões intergênicas do genoma de M. hyopneumoniae linhagem 7448. Dois tipos de repetições foram selecionados para a busca inicial: tandem e palindromes. Regiões intergênicas de até 500 pb a montante do sítio de início da tradução de todas as CDSs do genoma de M. hyopneumoniae linhagem 7448 foram utilizadas para a predição. Para cada tipo de elemento dois programas computacionais independentes foram utilizados. As predições in silico resultaram em 144 repetições em tandem e 1.171 palindromes. O DNA repetitivo se encontra distribuído a montante de 86% das unidades transcricionais de M. hyopneumoniae linhagem 7448. Análises comparativas entre genomas de micoplasmas demonstraram diferentes níveis de conservação dos elementos repetitivos entre linhagens patogênicas e não-patogênicas. Linhagens patogênicas revelaram uma conservação de 59%, enquanto que a não patogênica, somente de 46%. Através de ensaios de amplificação quantitativa de DNA, foi observado diferentes níveis de expressão em genes codificantes para importantes proteínas, como glicina hidroximetiltransferase, lipoproteína, adesinas e proteína ligadora de GTP. Os genes codificantes para essas proteínas divergiam no número de repetições palindromes e tandens na sua respectiva região intergênica. Além disso, repetições encontradas em 206 genes já descritos como regulados em diferentes condições em M. hyopneumoniae linhagem 232 mostraram aproximadamente 80% de conservação em relação à linhagem M. hyopneumoniae linhagem 7448. Todos esses resultados sugerem um potencial papel regulador das repetições de DNA em tandem e palindromes em Mycoplasma. / Mycoplasma hyopneumoniae is a diminutive bacterium, characterized by a small genome with a low GC content. It is commonly associated with swine respiratory diseases, resulting in productivity and economic losses in the animal industry. Repetitive DNA, which occurs in large quantities in eukaryotic cells, has been increasingly identified in prokaryotic genomes, and has been associated with a potential regulatory function. Once transcription regulation in these organisms is still poorly understood, the aim of the current study was to perform an in silico search of repeat elements in the genomic intergenic regions of M. hyopneumoniae strain 7448. Two types of repeats were selected for initial search: Tandem and Palindromic. Intergenic regions up to 500 bp upstream from start codon of M. hyopneumoniae strain 7448 CDSs were used as input for the software’s prediction. For each type of repeat sequence, two independent software packages were used. Computational analysis results in 144 tandem repeats and 1,171 palindrome elements. The repeats were distributed in the upstream region of 86% of transcriptional units of M. hyopneumoniae strain 7448. Comparative analysis between distinct mycoplasmas, demonstrate different indices of repeat conservation among pathogenic and non-pathogenic strains. Pathogenic strains revealed 59% conservation, while non-pathogenic only 46%. Through assays of quantitative amplification of DNA, different levels of expression in genes coding important proteins have been demonstrated, as glycine hydroxymethyltransferase, lipoprotein, adhesins and GTP-binding protein. These protein coding genes differ in number of palindromes or tandem repeats in respective upstream regions. In addition, repeats found in 206 genes already described to be regulated in different grow conditions in M. hyopneumoniae strain 232 showed almost 80% of conservation in relation to M. hyopneumoniae strain 7448. All these findings, suggests a potential regulatory role of tandem and palindrome DNA repeats.

No description available.

The ability of bacteria to detect their surroundings and enact an appropriate response is critical for survival. Translation of external signals into a coherent response requires specific control over the transcription of DNA into RNA. Much of the regulation at this step is accomplished by transcriptional regulators, proteins that bind to DNA and alter gene expression. A wide-spread variety of regulators in bacteria is the AraC-family. These regulators are divided into two conserved domains and respond to a variety of compounds owing to different N-terminal domains. A subfamily of these regulators, GATase1-like AraC-family transcriptional regulators (GATRs), is described. These proteins contain an N-terminal domain with structural characteristics similar to enzymes that synthesize amine-containing compounds. Members of this subfamily of transcriptional regulators are found in a wide range of bacteria, however, few are characterized. A relatively high number of GATRs are encoded in the Burkholderia thailandensis genome. Therefore, we utilized this bacterium as a model to explore the function and diversity of these regulators. GATRs in B. thailandensis divided into two groups based on bioinformatics analysis. The first group includes three members which we identified that contribute to the positive regulation of glycine betaine (GB) catabolism. GB can be utilized as a nutrient source or as a potent osmoprotectant. The regulation of this pathway in B. thailandensis differs from previously established models due to the interplay of these regulators. Homologs of two other GATRs in this group were identified that regulate carnitine and arginine catabolism. The second group of GATRs contains uncharacterized members with no known functions. A genetic strategy for engineering constitutive GATRs was developed and employed to investigate the transcriptional regulons of these GATRs. This approach yielded the identification of a novel GATR that represses expression of an operon producing a formaldehyde detoxification system, and is the first example of a GATR that functions as a repressor.

Pseudomonas aeruginosa RsmA belongs to the CsrA family of RNA binding proteins. CsrA family members are post-transcriptional regulators of global gene expression and usually function to inhibit translation of target genes, but in some cases can also exert positive regulatory effects. Previous work from our lab determined that RsmA is required for maximal T3SS gene expression in P. aeruginosa strain PA103. Nevertheless, the molecular mechanism underlying the RsmA-mediated control of T3SS gene expression was unknown. Expression of the T3SS is under the direct control of ExsA, a transcriptional activator. Previous microarray analyses showed that exsA transcript levels were reduced two-fold in an rsmA mutant. In chapter II I examine the role of RsmA in regulating ExsA expression. I demonstrate that expression of a ExsA-LacZ translational fusion was reduced two-fold in an rsmA mutant suggesting a specific effect of RsmA on ExsA expression. The effect of RsmA on ExsA expression occurs at a post-transcriptional level and is independent of mRNA and protein stabilization mechanisms. RsmA directly interacts with the exsCEBA transcript at multiple sites. Truncation analyses indicate that the -37 to +85 region (relative to the ATG start codon) is necessary and sufficient for RsmA-dependent control. I identified two binding sites, BS1 (-25 bp) and BS2 (+84), involved in the interaction of RsmA with the exsA transcript using sequence analysis, site-directed mutagenesis, EMSA assays, RNase footprints, and RNaseH cleavage assays. Mutagenesis of both binding sites results in an RsmA-independent phenotype. I further demonstrate that RsmA is able to activate ExsA expression. I propose a model wherein RsmA relieves a block on ExsA translation. Collectively, this work shows that RsmA directly binds and activates ExsA expression at the post-transcriptional level. Most Pseudomonas species carry at least two homologs of CsrA on the chromosome, but only one copy had been identified in P. aeruginosa. Through the course of other projects in the lab, we observed several phenotypes that could not be accounted for by a single copy of RsmA. In collaboration with the Wolfgang lab, we identified a second CsrA homolog, RsmF in P. aeruginosa. RsmF is dimeric in solution. The structure of RsmF differs substantially from other CsrA homologs by having alpha-helices located between the beta-2 and beta-3 strands. In chapter III I examine the role of RsmF in regulating RsmA-controlled processes associated with acute (T3SS) and chronic (T6SS and biofilm formation) infection. I discovered that while an rsmF mutant alone does not exhibit a phenotype, simultaneous deletion of both rsmA and rsmF significantly accentuates the phenotypes exhibited by an rsmA mutant alone. I show that RsmA directly binds and represses RsmF translation and that the small regulatory RNAs RsmZ and RsmY do not significantly modulate RsmF activity. Site-directed mutagenesis revealed that Arg 62, located in the beta-1 and beta-5 fold, is essential for biological activity in vivo and RNA-binding in vitro suggesting a conserved mechanism of RNA recognition maintained across all CsrA family members. Finally, I show that RsmF binds to only a subset of RsmA targets and is not involved in the regulation of all RsmA-controlled processes. In chapter IV I identified high-affinity RNA ligands from a chemically synthesized oligonucleotide library using systematic evolution of ligands by exponential enrichment (SELEX) and high-througput sequencing. From preliminary analyses of high-throughput sequencing data, the RsmF-binding consensus was determined as 5'-RUACARGGAC-3', with the ARGGA motif being 95% conserved. Collectively, this work shows that Rsm homologs play important roles in regulating virulence gene expression in P. aeruginosa.

The UDP-glucuronosyltransferases (UGTs) are a superfamily of enzymes that glucuronidate small, lipophilic molecules, thereby altering their biological activity and excretion. In humans, important examples of UGT substrates include molecules of both endogenous and xenobiotic origin; thus, UGTs are considered essential contributors to homeostatic regulation and an important defence mechanism against chemical insult. In keeping with both roles, UGTs are most strongly expressed in the liver, a predominant organ involved in detoxification. Rates of glucuronidation in humans are neither uniform among individuals, nor constant in an individual over time. Genetic determinants and non-endogenous signals are both known to influence the expression of UGTs, which in turn may affect the efficacy of certain pharmaceutical treatments or alter long-term risk of developing disease. Thus, this thesis focuses on the transcriptional regulation of UGT genes in humans, particularly on mechanisms that are likely to be relevant to their expression and variation in the liver. Two major approaches were used: firstly, extensive studies of several UGT promoters were performed to identify and characterise transcriptional elements that are important for UGT expression; and secondly, important hepatic transcription factors were investigated as potential regulators of UGT genes. UGT1A3, UGT1A4 and UGT1A5 are a subset of highly related, but independently regulated, genes of the human UGT1 subfamily. UGT1A3 and UGT1A4 are expressed in the liver, whereas UGT1A5 is not. The presented analysis of the UGT1A3, UGT1A4 and UGT1A5 proximal promoters demonstrates that a hepatocyte nuclear factor (HNF)1-binding site common to all three promoters is important for UGT1A3 and UGT1A4 promoter activity in vitro, but is insufficient to drive UGT1A5 expression. Two additional elements required for the maximal activity of the UGT1A3 promoter were also identified that may distinguish this gene from UGT1A4. UGT1A3 was investigated further, focusing on mechanisms that may contribute to interindividual variation in UGT1A3 expression. Polymorphisms in the UGT1A3 proximal promoter were identified and their functional consequences tested. Known variants of HNF1alpha were also tested for altered activity towards the UGT1A3 gene. UGT1A9 is the only hepatic member of the UGT1A7-1A10 subgroup of UGT1 enzymes. Previous work had identified HNF1-binding sites in all four genes, and HNF4alpha as an UGT1A9-specific regulator. The work presented herein extends these findings to show that HNF1 factors and HNF4alpha synergistically regulate UGT1A9, and that HNF4alpha is not the only transcription factor responsible for the unique presence of UGT1A9 in the liver. Liver-enriched transcription factors screened as potential UGT regulators were chosen from the HNF1, HNF4, HNF6, FoxA and C/EBP protein families. Functional interactions newly identified by this work were HNF4alpha with UGT1A1 and UGT1A6, HNF6 with UGT1A4 and UGT2B11, FoxA1 and FoxA3 with UGT2B11, UGT2B15 and UGT2B28 and C/EBPalpha with UGT2B17. Observations were also made regarding different patterns of interaction between each UGT and the transcription factors tested, particularly HNF1alpha.

Receptors that transport vitellogenin into oocytes are of vital importance to egg-laying species because they promote oocyte development. In this study, we describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA. Using reverse transcription polymerase chain reaction (RT-PCR) and both 5’- and 3’- rapid amplification of cDNA ends (RACE), cDNA fragments encompassing the entire coding region of a putative VgR from fire ant (= SiVgR) were cloned and sequenced. The complete SiVgR cDNA has a length of 5764 bp encoding a 1782-residue protein with a predicted molecular mass of 201.3 kDa. The deduced amino acid sequence of the SiVgR revealed that it encoded a protein belonging to the low-density lipoprotein receptor superfamily. The number and arrangement of modular domains of SiVgR are the same as those of mosquito and fruit fly VgRs, except there are only four Class A cysteine-rich repeats in the first ligand binding domain of SiVgR compared to five in the mosquito and fruit fly. The deduced amino acid sequence of the SiVgR exhibited 35% and 31% identity to those of the mosquito and fruit fly VgRs, respectively. Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in Northern blot analysis demonstrated that the 7.4-kb SiVgR mRNA was present only in ovaries of reproductive females − both alates (virgins) and queens (mated) and was more abundant in alates. The developmental profile of transcriptional expression was determined by semiquantitative RT-PCR. It showed that the SiVgR transcript increased 6-fold from 0- to 10-days after mating, then remained constant through 30 days. It also showed that the SiVgR transcripts increased with age in alate virgin females. The transcriptional expression of the SiVgR was up-regulated more than two-fold by methoprene, a juvenile hormone analog, as determined by using an in vitro system. This suggested the SiVgR gene is JH regulated.

La famille des protéines SIBLINGs comprend la sialoprotéine osseuse (BSP), lostéopontine (OPN), la sialophosphoprotéine de dentine (DSPP), la protéine de matrice de dentine 1 (DMP1), la phosphoglycoprotéine de matrice extracellulaire (MEPE) et lénameline (ENAM). Comme leur nom lindique, ces protéines ont dabord été identifiées au niveau de la matrice minéralisée de los et de la dent. Durant la dernière décennie, notre Laboratoire et dautres équipes ont démontré que cette famille de protéines est également exprimée par un certain nombre de tissus tumoraux. Nous avons entrepris ce doctorat dans la continuité des projets de recherche menés au Laboratoire sintéressant à létude des SIBLINGs au cours de la progression tumorale et métastatique. La première partie de notre projet a consisté à investiguer les mécanismes de régulation de lexpression du gène de la BSP humaine au niveau de cellules ostéoblastiques. Le facteur de transcription Runx2 est un facteur clé de la régulation des gènes osseux. Dès lors, nous avons émis lhypothèse selon laquelle linduction de lexpression du gène de la BSP observée au cours de la différenciation ostéoblastique pourrait être sous la dépendance de ce facteur. Nous avons ensuite étudié la régulation du gène de la BSP au niveau de cellules cancéreuses mammaires. En effet, nous avons voulu déterminer si lexpression de la BSP était sous la dépendance de mécanismes de régulation transcriptionnelle différents de ceux observés au niveau de cellules dorigine osseuse. Lobjectif final étant de bloquer spécifiquement lexpression de la BSP au niveau des tumeurs. Notre stratégie a dabord consisté à identifier les principaux facteurs transcriptionnels impliqués dans cette régulation puis à en étudier limpact sur lactivité du promoteur de la BSP au niveau des deux types cellulaires considérés. Dans la deuxième partie de ce projet, nous nous sommes consacrés à létude dun autre membre de la famille des SIBLINGs, lOPN, afin den identifier le rôle au niveau des gliomes humains. Précédemment, il a été démontré que lOPN est surexprimée dans les gliomes et ce, en corrélation avec le grade de la tumeur. Cependant, il ny a que peu détudes décrivant le rôle de lOPN dans les gliomes. Dès lors, nous avons voulu vérifier limportance de lOPN au cours du développement des gliomes en utilisant la technique dinterférence à lARN.

The proteasome is a major non-lysosomal proteolytic complex present in eukaryotic cells and has a central role in regulating many protein levels. The complex has been shown to participate in various intracellular pathways including cell cycle regulation or quality control of newly synthesized proteins and many other key pathways. This amazing range of substrates would not be possible without the help of regulators that are able to bind to the 20S proteasome and modulate its activity. Among those, the PA700 or 19S regulator and the PA28 family are the best characterized in higher eukaryotes. The 19S regulatory particle is involved in the recognition of ubiquitinated proteins, targeted for degradation by the proteasome. The PA28 (also termed 11S REG) family is composed of two members the PA28αβ and PA28γ. The function of PA28αβ is related to the adaptive immune response with a proposed contribution in MHC class I peptide presentation whereas the biological role PA28γ remains unknown. The main objectives of the laboratory, and subsequently of this thesis are to use Drosophila melanogaster model system and its advantages to better understand the precise contribution of these different activators in the regulation of the proteasome. Using genomic resources, a unique Drosophila PA28 member was identified, characterized and was shown to be a proteasome regulator with all the properties of PA28γ. Through site-directed mutagenesis we identified a functional nuclear localization signal in the homolog-specific insert region. Study of the promoter region revealed that transcription of Drosophila PA28γ (dPA28γ) gene is under control of DREF, a transcription factor involved in the regulation of genes related to DNA synthesis and cell proliferation. To confirm that dPA28γ has a role in cell cycle progression, the effect of removing dPA28γ from S2 cells was tested using RNA interference. Drosophila cells depleted of dPA28γ showed partial arrest in G1/S cell cycle transition confirming a conserved function between Drosophila and mammalian forms of PA28γ. Finally, characterization of the Dictyostelium regulator, an evolutionarily distant member of the PA28γ, was carried out using fluorogenic degradation assays. We are currently knocking-out the gene in order to determine the biological function of the activator. A second part of my work consisted in the generation of a Drosophila assay used to identify in vivo substrates of the 19S regulator, an assay system that has been originally engineered by Dantuma and coworkers in human cell lines. This was achieved by cloning of GFP behind a series of modified ubiquitins that create substrates degraded through different pathways involving the proteasome pathways. The last project of my thesis concerns the characterization of the mechanism for upregulation of proteasomal gene mRNA after MG132 (proteasome inhibitor) treatment. So far, we found that the 5´-UTR of the genes is responsible for this induction. We are now looking for the precise motif involved in this regulation.