Gliomas are the most common primary brain tumors and include astrocytomas, oligodendrogliomas and oligoastrocytomas. Astrocytomas have a high frequency of occurrence as compared to the other gliomas and several studies including ours have focused on understanding the etiology, biology and genetics of this disease. Based on the degree of malignancy, astrocytomas have been graded from I to IV. Grade I or pilocytic astrocytomas are benign tumors and have limited infiltration. On the contrary, Grade II-IV astrocytomas also referred to as diffusely infiltrating astrocytomas (DA, Grade II), anaplastic astrocytomas (AA, Grade III) and glioblastoma multiforme (GBM, Grade IV), have the tendency of diffusely infiltrating the normal brain parenchyma. GBM is characterized by uncontrolled proliferation and resistance to apoptosis, rampant invasion, recalcitrance to most established therapies etc which makes them the most aggressive of all gliomas with a median survival of about 12 months. This makes it imperative to initiate further studies to understand the molecular basis of this disease. Gene expression profiling studies have been central to this effort. In recent years, several Microarray studies have provided crucial insights into the biological role of novel genes not previously associated with astrocytomas. In a previous Microarray study, several differentially regulated genes in astrocytoma were identified in our laboratory. In addition to many up regulated genes, several down regulated genes were also identified in this study. Down regulated genes are interesting to study because of their relevance as possible tumor suppressor genes. Hence, we decided to characterize the regulation and functional significance of few down regulated genes. The specific objectives of the study are as follows 1)To validate novel down‐regulated genes in astrocytomas identified by a previous Microarray study. 2)To understand the mechanism of down-regulation of a few selected gene. 3)Functional characterization of DIRAS2, a novel astrocytoma down‐regulated gene with respect to its possible role in astrocytoma progression. Towards these objectives, we identified 21 genes as differentially down-regulated across all grades of astrocytoma based on a previous Microarray study from our lab and data from literature. Real time qRT-PCR analysis performed on these 21 genes confirmed their down-regulation in all grades of astrocytoma as compared to normal brain tissues. From these 21 genes, we short-listed 10 of the most consistently down-regulated genes for further analysis. These genes were DIRAS2, IGFBP9, MAL2, MBP, OLFM1, PACSIN1, RAB26, SYT1, SYT5 and VSNL1. We also confirmed the expression of two of the genes, OLFM1 and RAB26 at the protein level by performing immunohistochemical analysis on an independent set of 38 tissues that included 10 normal tissues and 28 tissues from different grades of astrocytoma. OLFM1 was found to be down-regulated in a grade specific manner. RAB26 expression was found to be strikingly high in all the low grade astrocytomas in comparison to high grade astrocytomas which made it an interesting gene to study functionally. On functional characterization, we found that RAB26 over‐expressing LN229 cells showed significantly reduced invasion compared to the vector transfected cells suggesting RAB26 could have a tumor-suppressing role in astrocytomas. In order to investigate whether transcriptional modulation could play a role in the down-regulation of these 10 genes, we searched for transcription factor binding sites in approximately 2kb 5’ flanking region of each gene. Intriguingly one or more PAX6 binding sites were present in all their promoters. In light of the fact that PAX6 has been proposed as a tumor‐suppressor in astrocytomas, we predicted that some of these genes could be targets of PAX6 transactivation and could possibly mediate some of the tumor‐suppressive actions of PAX6. PAX6 has been proposed as a down stream target of Notch signaling in the context of eye development. Similar to this observation, upon activation of Notch signaling with a virus expressing human intracellular domain of Notch (Ad-NIC-1), PAX6 expression was found to be induced in glioma cell lines suggesting PAX6 to be a novel NOTCH target in astrocytomas. In addition, Ad-NIC-1 infection could also induce the expression of OLFM1, RAB26, MAL2 and MBP in U343 cells. We could also demonstrate that Ad-NIC-1 co-operates with PAX6 in the regulation of these four genes in cell lines expressing endogenous PAX6, namely U343 and U251. Intriguingly, in a cell-line lacking PAX6 expression (LN229), Ad-NIC-1 could not induce OLFM1, RAB26 and MBP, although we could see induction of MAL2. Interestingly, PAX6 overexpression in LN229 cells in the absence of Ad-NIC-1 could induce OLFM1, RAB26 and MAL2. In contrast, infection of Ad-NIC-1 on the PAX6 over-expressing cells seemed to have an antagonistic effect on the expression of OLFM1, RAB26 and MBP, suggesting that Ad-NIC-1 antagonizes PAX6 actions in these cells. Ad-NIC-1 infection resulted in increased apoptosis in a PAX6 independent manner in U343 cells, which as previously mentioned has high levels of PAX6 endogenous expression. Conversely, Ad-NIC-1 could not induce apoptosis in LN229 cells, which has negligible expression of PAX6. We could also demonstrate that apoptosis induced in U343 cells could be in a p53 dependent manner. Activation of AMPK pathway and inhibition of the mTOR pathway as a consequence of p53 induction could also explain the Ad-NIC-1 mediated apoptosis that was seen in these cells. Thus, we have proposed that Notch signaling could possibly have a tumor-suppressing role in the presence of PAX6. We also suggest that down-regulation of OLFM1, RAB26, MAL2 and MBP via the NOTCH-PAX6 axis could be a possible molecular mechanism for the down-regulation of these genes. With respect to the third objective, we sought to characterize DIRAS2 with respect to its function in astrocytomas. DIRAS2 was identified as a down‐regulated gene in all grades of astrocytoma by our Microarray study. We were also able to validate the down‐regulation of DIRAS2 in all grades of astrocytomas. DIRAS2 also bears significant homology to RIG1 (also known as DIRAS1), which has been proposed as a tumor suppressor gene in astrocytomas. In the light of these data, we predicted that DIRAS2 could be a tumor suppressor gene in astrocytomas. Overexpression of DIRAS2 in two glioma cell lines U87 and C6 did not reveal any appreciable change in proliferation. Strikingly when the DIRAS2 over-expressing clones were grown in the absence of serum, there was marked increase in proliferation with respect to vector transfected clones along with a distinct change in morphology. Decorin expression in the DIRAS2 over-expressing clones was found to be up regulated and could be responsible for the altered morphology as well as enhanced viability in absence of serum. Interestingly along with Decorin expression, we also observed an increase in phosphor-SMAD2 levels indicative of activated TGF‐β signaling in the DIRAS2 over-expressing clones in the absence of serum. In the soft agar and migration/invasion assays, the results across the two cell lines, U87 and C6 were contrasting. DIRAS2 over-expressing clones of U87 cells formed visibly larger and increased number of colonies as compared to vector transfected clones and there was about a three fold increase in invasion with respect to that seen in vector transfected clones in the matrigel invasion assay. On the other hand, DIRAS2 over-expressing C6 clones formed colonies of smaller size compared to vector transfected clones and a marked decrease in migration was observed in the DIRAS2 over-expressing clones of C6. The discrepancies in the results in these two cell lines could be attributed to the presence of other regulators of DIRAS2 function unique to each of the two cell lines. Although in the present study, the results with respect to its predicted function as a tumor-suppressor gene has not been conclusive, the role of DIRAS2 in tumorigenesis may depend on the cellular context in which the protein is expressed. Overall in this study, we have identified a novel down regulated gene signature in astrocytomas consisting of OLFM1, RAB26, MAL2 and MBP. Furthermore, we have proposed that inhibition of NOTCH and PAX6 signaling pathways could be responsible for the down-regulated expression of OLFM1, RAB26, MAL2 and MBP in astrocytomas. Collectively, these results suggest that astrocytomas with activated Notch1 and/or Pax6 signaling could have good prognosis due to the tumor suppressive actions of OLFM1, RAB26, MAL2 and MBP

The chb operon constitutes the genes essential for utilization of chitooligosaccharides in Escherichia coli and related species. The six genes of the operon code for a transcriptional regulator (ChbR) of the operon, a permease (ChbBCA), a monodeacetylase (ChbG), and a phospho-beta-glucosidase (ChbF). In the absence of the substrate, the operon is maintained in a transcriptionally repressed state, while presence of the substrate leads to transcriptional activation. Regulation of the chb operon is brought about by the concerted action of three proteins, the negative regulator NagC coded by the nag operon, the dual function regulator ChbR coded by the chb operon and the universal regulatory protein CRP. Mutations that lead to alterations in the regulation of the operon can facilitate utilization of cellobiose, in addition to chitooligosaccharides by E. coli. The studies presented in Chapter II were aimed at understanding the evolution of cellobiose utilization in Shigella sonnei, which is phylogenetically very close to E. coli. Cel+ mutants were isolated from a Cel- wild type S. sonnei strain. Interestingly, Cel+ mutants arose relatively faster on MacConkey cellobiose agar from the S. sonnei wild type strain compared to E. coli. Similar to E. coli, the Cel+ phenotype in S. sonnei mutants was linked to the chb operon. Deletion of the phospho-β-glucosidase gene, chbF also resulted in loss of the Cel+ phenotype, indicating that ChbF is responsible for hydrolysis of cellobiose in these mutants. Previous work from the lab has shown that acquisition of two classes of mutations is necessary and sufficient to give rise to Cel+ mutants in E. coli. The first class of mutations either within the nagC locus or at the NagC binding site within the chb promoter, lead to NagC derepression. The second class consisting of gain-of-function mutations in chbR enable the recognition of cellobiose as an inducer by ChbR and subsequent activation of the operon. However, in S. sonnei a single mutational event of an IS element insertion resulted in acquisition of this phenotype. Depending on the type and location of the insertion, the mutants were grouped as Type I, and Type II. In Type I mutants an 1S600 insertion between the inherent -10 and -35 elements within the chb promoter leads to ChbR-independent constitutive activation of the operon, while in Type II mutants, an IS2/600 insertion at -113/-114, leads to ChbR-dependent, cellobiose-inducible expression of the operon. The results presented also indicate that in addition to relieving NagC mediated repression, the insertion in Type II mutants also leads to increase in basal transcription from the chb promoter. Constitutive expression of the chb operon also results in utilization of the aromatic β-glucosides salicin and arbutin, in addition to cellobiose in Type I mutants, which indicates the promiscuous nature of permease and hydrolysis enzyme of the chb operon. This part of the thesis essentially demonstrates the different trajectories taken for the evolution of new metabolic function under conditions of nutrient stress by two closely related species. It emphasizes the significance of the strain background, namely the diversity of transposable elements in the acquisition of the novel function. The second part of this research investigation, detailed in Chapter III deals with experiments to characterize the eukaryotic orthologs of the last gene of the chb operon. The chbG gene of E. coli codes for a monodeacetylase of chitooligosaccharides like chitobiose and chitotriose. The protein belongs to a highly conserved, but less explored family of proteins called YdjC, whose orthologs are present in many prokaryotes and eukaryotes including mammals. The human YDJC locus located on chromosome 22 is linked to a variety of inflammatory diseases and the transcript levels are relatively high in stem cells and a few cancer cells. In silico analysis suggested that the mammalian YdjC orthologs possess sequence and structural similarity with the prokaryotic counterpart. The full length mouse YdjC ortholog, which is 85% identical to the human ortholog was cloned into a bacterial vector and expressed in a chbG deletion strain of E. coli. The mouse YdjC ortholog could neither promote growth of the strain on chitobiose nor induce transcription from the chb promoter. The purified mouse YdjC ortholog could not deacetylate chitobiose in vitro as well, suggesting that the mouse ortholog failed to complement the function of the E. coli counterpart, ChbG under the conditions tested in this study. In order to characterize the mammalian YdjC orthologs more elaborately, further experimentation was performed in mammalian cell lines. The results indicate that YdjC is expressed in mammalian cell lines of different tissue origin and the expression was seen throughout the cell. Overexpression of mouse Ydjc in a few mammalian cells also resulted in increased proliferation and migration, indicating a direct or indirect role of this protein in cell growth/proliferation. The mammalian orthologs of ChbG therefore appear to have related but distinct activities and substrates compared to the bacterial counterpart that need to be elucidated further.

La régulation des gènes est à l’origine de la diversité cellulaire en permettant aux cellules de se différencier et de se spécialiser. La régulation génique repose largement sur l’existence de séquences d’ADN non codantes dans le génome, appelées "éléments cis-régulateurs", qui vont permettre de recruter de nombreux facteurs de transcription afin de former d’importants complexes (nucléo)protéiques qui vont agir sur le niveau de transcription des gènes. Ce recrutement est notamment contrôlé par des modifications épigénétiques. Le développement des techniques de séquençage et des méthodes d’analyse bioinformatiques permettent d’intégrer de grandes quantités de données pour étudier le fonctionnement des éléments régulateurs. Dans un premier temps, l’intégration de l’ensemble des données ChIP-seq disponibles dans les bases de données nous a permis de créer un catalogue d’éléments régulateurs putatifs chez l’Homme. L’analyse de ce catalogue nous a alors mené à caractériser ces éléments et à mettre en évidence la complexité combinatoire des facteurs de transcription. Dans un deuxième temps, nous avons réalisé une étude basée sur l’analyse des éléments régulateurs impliqués dans la différenciation précoce des lymphocytes T chez la souris. Cette étude a permis de mettre en évidence deux niveaux de complexité impliqués dans la régulation des gènes : le premier est basé sur la combinatoire des facteurs de transcription au sein des éléments régulateurs et le second repose sur la combinatoire des éléments eux-mêmes. Finalement, nous avons développé une nouvelle technique d’analyse quantitative et à haut débit de l’activité régulatrice de régions génomiques chez les mammifères. / Gene regulation is responsible for cell diversity by allowing cell differentiation and specialisation. Gene expression regulation relies mainly on the existence of non-coding DNA sequences in the genome, called "cis-regulatory elements", which recruit numerous transcription factors to form (nucleo)protein complexes which act on the gene transcription level. This recruitment is controlled in particular by epigenetic modifications. The rapid development of sequencing technologies and bioinformatics methods makes possible the integration of large amounts of data to study regulatory elements. First, the integration of ChIP-seq data for all transcription factors available in public databases has allowed us to create an extensive catalogue of putative regulatory elements in the human genome. The overall analysis of this catalogue led us to further characterize these elements and to highlight the high level of combinatorial complexity of transcription factors in the genome. Secondly, we conducted a more specific study based on the analysis of the regulatory elements involved in the early differentiation of T-cells in mice. This study provided an opportunity to highlight two levels of complexity based on regulatory elements and involved in gene regulation: the first rests on the transcription factor combinatorial in regulatory elements and the second is based on the combinatorial of elements themselves within loci. Finally, to validate experimentally the regulatory elements, we have developed a new quantitative and high-throughput technique to assess the regulatory activity of genomic regions in mammals.

Em bactérias, os fatores sigma alternativos permitem a rápida adaptação da célula a alterações no ambiente. Dentre estes, os fatores sigma ECF (função extra-itoplasmática) caracterizam-se pelo envolvimento na resposta a sinais da região extra-citoplasmática da célula. O sequenciamento do genoma de Caulobacter crescentus indicou a presença de 13 ORFs codificando fatores ECF. Este trabalho descreve a caracterização de cepas mutantes em cinco genes que codificam fatores sigma ECF de C. crescentus, sendo eles sigL, sigM, sigN, sigU e sigF. A regulação da expressão destes genes em respostas a diferentes estresses foi também analisada, pelo uso de fusões de transcrição de suas regiões promotoras ao gene reporter lacZ. Todos os mutantes mostraram-se viáveis, sendo também tão resistentes quanto a cepa parental a uma série de estresses ambientais testados, indicando que estes genes não são essenciais. Verificou-se, porém, que os mutantes nos genes sigL e sigM são mais sensíveis ao choque térmico extremo (48°C). A caracterização da cepa mutante em sigF mostrou que este gene é essencial para a resistência da célula a estresse oxidativo durante a fase estacionária. A análise da expressão de sigF indicou um controle pós-transcricional, com o acúmulo da proteína SigF durante esta fase do crescimento, sem um aumento na transcrição do gene. Oito genes regulados por &#963;F durante a fase estacionária foram identificados em experimentos de \"microarray\", incluindo os genes de resposta a estresse oxidativo, sodA e msrA. A análise da atividade do promotor de sigU mostrou sua indução na entrada na fase estacionária e após estresse salino e osmótico. No entanto, o mutante nulo em sigU não se apresentou mais sensível que a cepa parental a esses estresses. Os resultados aqui descritos permitiram identificar a importância de alguns dos fatores ECF de C. crescentus na resposta a estresses nesta bactéria. / Alternative sigma factors permit the rapid adaptation to environmental changes in bacteria. Among them, members of the ECF subfamily (extrac</i<ytoplasmic function) are characterized by their involvement in responses to changes in the extracytoplasmic compartment of the cell. Analysis of the complete genome sequence of Caulobacter crescentus has led to the identification of 13 ORFs encoding putative sigma factors of the ECF subclass. The present work describes the characterization of mutant strains in five genes encoding ECF sigma factors from C. crescentus, named sigL, sigM, sigN, sigU and sigF. The expression of these genes in response to distinct stress conditions was also investigated, using transcriptional fusions of their promoter regions to the lacZ reporter gene. The five mutants strains obtained were viable and did not show increased sensitivity, when compared to the parental strain, to a series of environmental stress conditions, indicating that these genes are not essential. However, the sigL and sigM mutant strains were shown to be more sensitive to extreme heat shock (48°C). Furthermore, the characterization of the sigF mutant strain demonstrated that this gene is essential for oxidative stress survival during stationary phase. Analysis of sigF expression indicated a post-transcriptional control, with an increase in the levels of SigF protein during this growth phase, without changes in the transcription rate of the gene. Eight genes regulated by &#963;F during stationary phase were identified in microarray experiments, including the oxidative stress response genes sodA and msrA. Analysis of sigU promoter activity in response to distinct stress conditions showed induction upon entry into stationary phase and during saline and osmotic stress. Nevertheless, the sigU null mutant did not show increased sensitivity to these stresses. The results described here identified the importance of some of the C. crescentus ECF sigma factors in the response to stresses in this bacterium.

O ciclo de divisão celular compreende uma seqüência de fenômenos controlados por una complexa rede de regulação gênica muito estável e robusta. Aplicamos as Redes Genéticas Probabilísticas (PGNs) para construir um modelo cuja dinâmica e robustez se assemelham às observadas no ciclo celular biológico. A estrutura de nosso modelo PGN foi inspirada em fatos biológicos bem estabelecidos tais como a existência de subsistemas integradores, realimentação negativa e positiva e caminhos de sinalização redundantes. Nosso modelo representa as interações entre genes como processos estocásticos e apresenta uma forte robustez na presença de ruido e variações moderadas dos parâmetros. Um modelo determinístico recentemente publicado do ciclo celular da levedura não resiste a condições de ruido que nosso modelo suporta bem. A adição de mecanismos de auto excitação, permite a nosso modelo apresentar uma atividade oscilatória similar à observada no ciclo celular embrionário. Nossa abordagem de modelar e simular o comportamento observado usando mecanismos de controle biológico conhecidos fornece hipóteses plausíveis de como a regulação subjacente pode ser realizada na célula. A pesquisa atualmente em curso procura identificar tais mecanismos de regulação no ciclo celular da levedura, usando dados de expressão gênica provenientes de medições seqüenciais de microarray. / The cell division cycle comprises a sequence of phenomena controlled by a stable and robust genetic network. We applied a Probabilistic Genetic Network (PGN) to construct an hypothetical model with dynamical behaviour and robustness typical of the biological cell-cycle. The structure of our PGN model was inspired in well established biological facts such as the existence of integrator subsystems, negative and positive feedback loops and redundant signaling pathways. Our model represents genes\' interactions as stochastic processes and presents strong robustness in the presence of moderate noise and parameters fluctuations. A recently published deterministic yeast cell-cycle model collapses upon noise conditions that our PGN model supports well. In addition, self stimulatory mechanisms can give our PGN model the possibility of having a pacemaker activity similar to the observed in the oscillatory embryonic cell cycle. Our approach of modeling and simulating the observed behavior by known biological control mechanisms provides plausible hypotheses of how the underlying regulation may be performed in the cell. The ongoing research is lead to identify such regulation mechanisms in the yeast cell-cycle from time-series microarray gene expression data.

P. aeruginosa PA14 é uma linhagem isolada de queimadura que apresenta vários fatores de patogenicidade comuns no quadro de infecção de hospedeiros filogeneticamente distintos (plantas, mamíferos ou invertebrados). O gene kerV foi revelado numa busca por mutantes atenuados em virulência em uma biblioteca de mutantes por transposons da linhagem PA14 (Rahme et al., 1997). A caracterização da linhagem D12, mutante em kerV, confirmou sua virulência atenuada (Apidianakis et al., 2005 e An et al., 2009) e resultados do transcriptoma mostraram alteração na expressão de mais de 500 genes, sendo alguns relacionados com o sistema de \"quorum sensing\" (Rahme et al, dados não publicados). O gene kerV está próximo à montante ao gene gloB, envolvido em detoxificação de metilglioxal, e à jusante aos genes rnhA e dnaQ, que codificam proteínas envolvidas na replicação e reparo do DNA. Este trabalho teve como objetivo estudar a função molecular do produto de kerV e a expressão dos genes do lócus kerV-rnhA-dnaQ. Análises de bioinformática indicam que a proteína KerV é uma metiltransferase dependente de S-adenosil-metionina (SAM), apresentando um domínio conservado de ligação a SAM e uma arquitetura de domínio compatível com a organização em fitas-beta e hélices-alfa alternadas descritas para a família das metiltransferases dependentes de SAM. Ela não apresenta outros domínios conservados que indiquem seu substrato de metilação. A expressão heteróloga desta proteína em E. coli, mostrou que ela é expressa de maneira parcialmente solúvel quando co-expressa com as chaperoninas GroEL/GroES em baixas temperaturas ou quando fusionada a MBP ou GST. A purificação desta proteína mostra que ela é co-eluída com a chaperonina GroEL sugerindo que para atingir sua conformação nativa ela necessita dessas proteínas acessórias. MBP-KerV purificado foi usado para ensaios \"in vitro\" de atividade de metiltransferase e ligação a SAM, que não foram conclusivos, pois não há certeza do seu correto estado de enovelamento. Ensaios de duplo-híbrido mostraram que KerV não interage com os produtos de rnhA e dnaQ, sugerindo que KerV não está diretamente relacionado com suas funções. A freqüência de mutação na linhagem D12 está levemente aumentada (aproximadamente quatro vezes), o que sugere que KerV não está diretamente envolvida no reparo de DNA do tipo ´mismatch repair`. Os ensaios usados para detectar metilação do DNA, proteínas e rRNAs não revelaram que KerV estaria envolvido com a metilação destes substratos. Os inícios de transcrição dos genes kerV, rnhA e dnaQ foram determinados. A deleção de kerV causa um efeito polar na transcrição do gene rnhA, que não se reflete nos níveis da proteína. A deleção também afeta a expressão de dnaQ, sugerindo que KerV seja importante para sua regulação. Os ensaios de complementação da virulência em modelos invertebrados e de células epiteliais de pulmão mostram que apenas a presença dos três genes e seus produtos em níveis normais são capazes de reverter a maioria dos fenótipos atenuados. KerV se mostrou essencial para a inibição da translocação de NF-kB para o núcleo das células, comprovando que esta proteína é relevante para a virulência de PA14, contribuindo com o silenciamento da resposta imune do hospedeiro. O conjunto dos resultados indicam uma complexa inter-relação entre a expressão dos genes kerV, rnhA e dnaQ e seu papel na biologia de P. aeruginosa. / P. aeruginosa PA14 is a burn isolate multi-host pathogen strain. The screening for virulence attenuated mutants in a PA14 transposon mutant library revealed the kerV gene (Rahme et al., 1997). The characterization of D12 strain, a kerV mutant, confirmed the attenuated virulence phenotype (Apidianakis et al., 2005 and An et al., 2009) and transcriptome analysis showed the expression of more than 500 genes are affected in D12, some of these genes are related with quorum sensing (Rahme et al, unpublished data). kerV is upstream of the gloB gene, related with methylglioxal detoxification and downstream of the rnhA and dnaQ genes, both related with DNA replication and repair. The purpose of this work was to study the molecular function of KerV product and the expression of kerV-rnhA-dnaQ locus. Bioinformatics analysis indicated that KerV is a SAM dependent methyltransferase that have a conserved SAM binding domain with architecture compatible with classic alternating &#946;-stranded and &#945;-helical regions. KerV does not show any other conserved motif that could indicate its methylation substrate. Heterologous expression in E. coli showed that KerV is partially soluble only when co-expressed with GroeL/GroES chaperones at low temperatures or when KerV is in fusion with MBP or GST tag. During the purification process KerV was copurified with GroEL chaperone suggesting that this association may be required for the correct folding of KerV. Methyltransferase activity and SAM binding assays were done with purified MBPKerV and the results were not conclusive since the proper conformation of MBP-KerV cannot be verified. Yeast two-hybrid assays indicated that RNaseH and DnaQ are not interaction partners of KerV, suggesting that their functions are not directly related. The mutation frequency of D12 strain increased only about four times in relation to PA14, suggesting that KerV is not directly involved with DNA mismatch repair. The assays to detect methylation in DNA, RNAs and proteins do not show that KerV is involved with methylation of these substrates. The transcription start sites of kerV, rnhA and dnaQ genes were mapped through 5\'-RACE- and primer extension experiments. The kerV deletion causes a polar effect on the transcription of rnhA gene, which is not reflected on RNaseH protein levels. The kerV deletion also affects dnaQ expression, suggesting that KerV is important for its regulation.The virulence complementation assays in flies and lung epithelial cells showed that the fully rescue of the wild type phenotype was achieved only when the entire locus is present. KerV was essential to inhibit the NF-kB nucleus translocation, demonstrating that KerV is relevant to PA14 virulence, contributing for the silencing of host immune system. Altogether, these data showed a complex inter-relation among kerV, rnhA and dnaQ genes and its role in P. aeruginosa biology

Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que pode se comportar como um patógeno oportunista. A linhagem PA14 apresenta duas ilhas de patogenicidade. A maior delas, PAPI-1, contém dois grupos de genes envolvidos com virulência, transcritos de maneira oposta e que estão entre duas seqüências repetidas diretas. O primeiro grupo compreende quatro genes dispostos em dois operons, que codificam para proteínas de sistemas de dois componentes (PvrS, PvrR, RcsC e RcsB). PvrS e RcsC são proteínas sensoras híbridas, que apresentam domínios de histidina-quinase e de reguladores de resposta. PvrR é um regulador de resposta com um domínio EAL com atividade de fosfodiesterase de diGMP cíclico e RcsB apresenta um domínio de ligação a DNA, além de um domínio fosfoaceptor. O outro grupo é composto de cinco genes, cupD1 a cupD5, que codificam para uma fímbria do tipo chaperone-usher e que apresenta alta similaridade com cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Trabalhos anteriores mostraram que pvrS, pvrR, rcsC, rcsB e cupD2 estão relacionados com a virulência de PA14. Como estes grupos de genes parecem ter sido inseridos na ilha em um único evento de recombinação, este trabalho investigou se os sistemas de dois componentes estão relacionados com a regulação da expressão de cupD. Foi observado que a expressão de cupD é maior a 28ºC do que que a 37ºC e é influenciada positivamente pelo regulador global de expressão, MvaT, uma proteína tipo H-NS. Ensaios de &#946;-galactosidase a partir de uma fusão de transcrição mostraram que a atividade promotora de cupD é cerca de 50% menor numa linhagem com deleção em rcsB em relação à linhagem selvagem. Nenhuma diferença consistente foi observada entre as linhagens com deleções em pvrS, pvrR, rcsC e rcsB e PA14 em relação a motilidade dos tipos swarming, swimming ou twitching ou à formação de biofilme. A linhagem de P. aeruginosa PA14 superexpressando RcsB mostrou níveis exacerbados de mRNA de cupD1, sendo a atuação de RcsB específica em cupD, já que os outros grupos de genes cup presentes em PA14 não mostraram a mesma variação na expressão, conforme analisado por RT-PCR quantitativo. Essa linhagem mostrou também um aumento na formação de biofilme, sem que a motilidade fosse alterada. Ainda visando elucidar os mecanismos de regulação de cupD, linhagens que superexpressam pvrR também foram analisadas quanto a estes fenótipos. Nesse caso, a superexpressão de pvrR diminuiu a formação de biofilme, conforme esperado, aumentou a motilidade do tipo swarming, porém não alterou a expressão de cupD. Os dados do presente trabalho demonstraram que a cupD é regulado pelos genes do sistemas de dois componentes adjacentes a ele e que o ativador de transcrição RcsB está relacionado com a formação de biofilme em tubos de vidro, provavelmente via a fímbria CupD. / Pseudomonas aeruginosa is a &#947;-proteobacteria that can behave as an opportunistic pathogen. The strain PA14 carries two pathogenicity islands, the largest of them, PAPI-1, contains two gene clusters between two direct repeat sequences that are transcribed in opposite directions and are involved in virulence. The first group consists of four genes arranged in two operons encoding two-component system proteins (PvrS, PvrR, RcsC and RcsB). PvrS and RcsC are hybrid sensor proteins, which contain domains of histidine kinase and response regulator domains. PvrR is a response regulator with a phosphodiesterase EAL domain and RcsB presents a C- terminal HTH DNA biding domain, in addition to a phosphoaceptor domain. The other group is composed of five genes, cupD1-5, that encodes components and assembly factors of a putative fimbrial CupD, which has high similarity with CupA, involved in the biofilm formation in other P. aeruginosa strains. Earlier work showed that pvrS, pvrR, rcsC, rcsB and cupD2 are related to the virulence of PA14. As these groups of genes appear to have been inserted on the island in a single event of recombination, this study investigated whether the two-component systems are related to the regulation of cupD expression. It was observed that cupD promoter activity is higher at 28oC than at 37oC and it is positively influenced by the global regulator, MvaT, a H-NS like protein. A lacZ transcriptional fusion showed about 50% less promoter activity of cupD from a strain with deletion in rcsB as compared to PA14. No consistent differences were found among the strains with deletions in pvrS, pvrR, rcsC and rcsB and PA14 on swarming, swimming and twitching motilities or biofilmsformation. A strain overexpressing overexpression showed heigher levels of cupD1mRNA of, and the role of RcsB as an activator is specific to cupD, as the other groups of cup genes present in PA14 did not show the same variation in the expression, as analyzed by quantitative RT-PCR. This strain also showed an increase in biofilm formation. In further assays aiming to elucidate the mechanisms of regulation of cupD, a strains overexpressing pvrR was also analyzed. Overexpression of pvrR decreased the formation of biofilm, as expected, and increased swarming motility, but did not alter the expression of cupD. The data from this study demonstrated that cupD is regulated by RcsB, and that this transcriptional activator is involved in the formation of biofilm in glass tubes, probably via CupD fimbriae.

Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.

La progression du cancer du sein repose principalement sur la signalisation des œstrogènes et de la progestérone, et les traitements modulant l’action des œstrogènes ont amélioré la survie des patientes atteintes d’un cancer à récepteurs œstrogéniques (ERα). Des études récentes convergent sur le concept selon lequel, dans les cancers du sein ER+, PR (Progesterone Receptor) peut inhiber les fonctions favorisant la croissance induite par l'œstrogène en reprogrammant directement la liaison d'ERα sur de nouveaux gènes cibles. Les données cliniques montrent que cette signature génique est associée à un bon pronostic dans une cohorte de 1.959 patientes atteintes de cancer du sein et qu’un agoniste de la progestérone améliore l'activité antiproliférative des thérapies anti-oestrogéniques1. Ainsi, ces données démontrent qu’ER n’est pas le seul acteur de la tumorigénèse mammaire et qu'il existe une interférence fonctionnelle entre ces deux voies hormonales, soulignant le besoin d’une meilleure compréhension de la signalisation de PR. D’un point de vue mécanistique, l’activité de PR est étroitement liée à l’interaction avec les nucléosomes. En effet, PR fonctionne comme un facteur « pionnier » et se lie à la chromatine au sein de complexes protéiques, régulant son activité transcriptionnelle. Sans progestérone, PR forme un complexe répressif associé à des enzymes modificatrices de la chromatine comme LSD1, HDAC1/2 et la protéine de l'hétérochromatine HP1γ2. En réponse au traitement hormonal, ce complexe est déplacé, ce qui permet de recruter des coactivateurs et des cofacteurs associés, qui modifient la structure de la chromatine locale et entraînent l'activation ou la répression des gènes cibles de PR. Nous avons identifié un nouveau régulateur de la signalisation de la progestérone, l'arginine méthyltransférase PRMT1, enzyme souvent surexprimée dans les cancers mammaires3,4. Par diverses approches in vitro et in vivo, nous avons montré une interaction directe entre PR et PRMT1, dans le noyau des cellules tumorales mammaires, et à la fois en absence d’hormone et après 1h de stimulation à la progestérone. De plus, PRMT1 apparaît comme un nouveau membre du complexe répressif sur la chromatine, associé à PR et à ses partenaires, dans un sous-ensemble de gènes inductibles par la progestérone. Nos résultats indiquent également que l’expression de PRMT1 affecte l’activité transcriptionnelle de PR et que son inhibition perturbe l’activation rapide de la voie de la protéine kinase après une stimulation progestative. Nous montrons pour la première fois que PR est méthylé sur un résidu arginine, conservé parmi les récepteurs nucléaires (R637), localisé dans son domaine de liaison à l'ADN. La production d’un anticorps dirigé contre la forme méthylée de PR nous a permis de préciser qu’elle se localisait dans le noyau des cellules et n’était retrouvée qu’après traitement progestatif. En outre, la mutation de R637 de PR entraine une diminution de l’expression d'un sous-ensemble de cibles de PR, ce qui entraine un retard de croissance cellulaire. En conclusion, ces résultats confirment l'implication de PRMT1 et de son activité méthyltransférase dans la signalisation de PR et plus particulièrement dans son activité transcriptionnelle. Nous démontrons donc que la méthylation sur résidus d'arginine est un nouveau mécanisme de contrôle lors de la réponse à la progestérone dans les cellules tumorales mammaires / Breast cancer progression is mainly driven by estrogen and progesterone signalling and therapies modulating oestrogen‘s action have improved the survival of ER+ cancer patients. As progesterone receptor (PR) is an ER target gene, its expression in breast cancer was considered as a predictive marker of ER functionality. However, recent studies are converging on the concept that PR can directly affect ER functions in breast cancer cells1. Activated PR can redirect ER to novel chromatin binding sites associated with cell differentiation and apoptosis, leading to a potential improvement of the tumour response to anti-oestrogen therapies. In considering the differential effects of progesterone in breast cancer, it is important to define the variable might influence progesterone pathway and the downstream mediators involved in this signalling. Recently, Beato and al reported that, in breast cancer cells, the unliganded form of PR (non-activated with progesterone) bind to genomic sites and target a repressive complex containing enzyme modifying chromatin as the demethylase LSD1 or the Heterochromatin Protein 1 (HP1γ)2. Under hormonal treatment, this complex is displaced, which makes it possible to recruit coactivators and associated cofactors, which modify the structure of the local chromatin and cause the activation or repression of the target genes of PR. In addition, cellular response to progesterone is also regulated by receptor post-translational modifications that may affect its stability, its subcellular localization and its interactions with regulators. In our study, we demonstrated for the first time that PR is methylated on arginine residues, by the arginine methyltransferase PRMT1. We identified as target the arginine 637 (R637), a conserved arginine among nuclear receptor superfamily, located in the DNA-binding domain of the receptor. By in vitro and in vivo approaches, we are studying the impact of PRMT1 on PR signalling pathways. In T47D breast cancer cells, we demonstrated that PR interacts with PRMT1, mainly in the nucleus. Of interest, PRMT1 interacts with PR in the nucleus in absence of hormone stimulation and it appears as a new member of the repressive complex on a subset of progesterone inducible genes. Our results also indicate that PRMT1 expression affects PR transcriptional activity and PRMT1 knockdown disrupts the rapid activation of protein kinase pathway after progestin stimulation. The production of an antibody directed against the methylated form of PR allowed us to specify that methylated-PR is localized in the nucleus of cells and was found only after progesterone treatment. Furthermore, PRMT1 depletion and mutation of R637 resulted in an inhibition of a subset of PR-regulated genes which led to retarded cell growth.Our data reveal the impact of PRMT1 expression on PR pathways and provide evidence for the asymmetric arginine dimethylation of PR. We therefore demonstrate that methylation on arginine residues could be a novel control mechanism in the response to progesterone in mammary tumor cells

<p>In recent years, science has shifted from a focus on reductionist explanations of biological phenomena to a more integrated, systems approach. This shift has made its way into curricular recommendations for undergraduate education. To understand complex biological phenomena, it has been argued that students employ mechanistic reasoning, in which one describes a mechanism by identifying the activities that produce change, the entities which engage in activities, and the starting and ending conditions. Students’ use of mechanistic reasoning requires engaging in the complex task of simultaneously integrating and coordinating multiple elements across space and time. In addition, students must link and organize their scientific ideas and then structure their thoughts into a network of knowledge, as described by the theory of knowledge integration. Previous studies that have explored students’ understanding of scientific concepts using knowledge integration as a lens found that students’ nonmechanistic ideas prevented them from identifying gaps in the connections between their ideas and from forming normative knowledge. Thus, this dissertation investigates the features of undergraduate biology students’ systems thinking using knowledge integration and mechanistic reasoning as conceptual and analytical frameworks. Using a semi-structured interview, we asked students to describe functional definitions of and relationships between three fundamental modules in biology (i.e., gene regulation, cell-cell communication, and the relationship between genotype and phenotype). In the first study, we found that the majority of students did not have normative functional definitions for how and why gene regulation occurs or how phenotype is regulated. When describing the relationships in an open context, most students expressed unidirectional, linear knowledge networks which lacked Mechanistic connections. In our second study, we examined how students described a transition point in biofilm development after being cued to think about the three modules. Though students struggled to transfer over relevant ideas to the biofilm context (such as gene regulation and cell-cell communication processes), we found that explanations were more specified in the nature of connections and content including more Mechanistic descriptions. In the third study, we explored features of biology students’ and instructors’ knowledge networks in an open context and situated to a context of the participants’ choice. Within an open context, most students described multidirectional, non-linear knowledge networks similar to instructors. In the specific context, the majority of students described non-linear knowledge networks, but some students modified their structures to be linear. Although the structures became less complex in the specific context, the nature of connections and content became more specified. Across all studies, we found that context greatly affected students’ systems thinking as revealed by the changing features of the knowledge networks. Specifically, context helped students identify what relationships they deeply understood and could transfer and allowed for the creation of a detailed explanation relevant to the specific biological phenomenon. For students to develop a broad systems perspective of biology, we recommend instructors engage students in the process of knowledge integration. Embed opportunities for students to think about biology concepts in various contexts, particularly where students grapple with nuanced and complex transfer of ideas. These practices will encourage students to form causal, mechanistic linkages between concepts and build an integrated, expert-like understanding of biology.</p>