Os genes de choque térmico groES e groEL de Caulobacter crescentus foram isolados utilizando-se os genes homólogos de E.coli como sonda e por sequenciamento demonstrou-se que estes genes estão organizados na forma de um operon em um fragmento de DNA de aproximadamente 2,5 kb, contendo também sua região regulatória. \"Northern blots\" de RNA total de células crescidas a 300C ou submetidas a choque térmico mostraram a presença de um único RNA de tamanho aproximado de 2,3kb, altamente induzido por choque térmico, permanecendo em altos níveis mesmo após longos períodos de choque térmico. Amostras de RNA total de células sincronizadas, de diferentes estágios do ciclo celular de Caulobacter, foram também analisadas mostrando que os níveis do mRNA groESL variam durante o ciclo, apresentando um máximo na célula prédivisional. Análises através de \"Western blot\" mostraram uma pequena variação nos níveis da proteína GroEL ao longo do ciclo celular, sendo os tempos 60 e 120 minutos, respectivamente, os pontos de mínimo e máximo acúmulo da proteína concordando com os resultados obtidos em \"N orthern blots\". O mesmo tipo de análise foi feito com extratos totais obtidos a partir uma população mista de células crescidas a 300C e submetidas a choque térmico, observando-se o acúmulo da proteína até 60 minutos depois do choque térmico, com aumento da ordem de 5 vezes nos níveis de GroEL, níveis estes que diminuem lentamente a partir deste ponto. Os inícios de transcrição foram determinados em experimentos de \"primer extension\" utilizando-se RNA total de células incubadas 300C e de células submetidas a diferentes condições de choque térmico. Dois possíveis sítios de início de transcrição foram determinados nas posições -119 e -88 do ATG da metionina iniciadora de groES, sendo as regiões -10 e -35 dos promotores correspondentes (P 1 e P2) identificadas. Somente a transcrição iniciando a partir de P2, que apresenta características de um promotor transcrito pelo σ32, aumenta durante o choque térmico. Fusões de transcrição com o vetor repórte placZ/290 e a região 5\' regulatória do operon groESL foram construídas para identificar as sequências responsáveis pelo controle por choque térmico e pelo controle temporal. Fusões de transcrição contendo deleções na região 5\' do operon mostraram que sequências a montante do promotor P2 não são necessárias para a indução por choque térmico ou para o controle temporal. Fusões de transcrição contendo mutações sítio-dirigidas na repetição invertida, encontrada a 3\' do promotor P2, antes do gene groES, revelaram que este elemento, conhecido como CIRCE, regula negativamente a expressão de groESL a 300C e mutações neste elemento levam à perda do controle temporal deste operon. / The heat shock genes groES and groEL of Caulobacter crescentus were isolated using the homologous genes of E.colí as a probe. DNA sequence analysis has shown that these genes are organized as an operan in a fragment of about 2.5kb, which includes the 5\' regulatory region. Northern blot analysis of total RNA from cells grown at 300C or heat shocked treated has shown the presence of a single mRNA species for groESL, of approximately 2.3kb in size, which presented increased leveis even after long periods of heat shock. Samples of total RNA from synchronized cells, corresponding to different stages of the Caulobaaer cell cycle, were also analysed, showing that the amount of groESL mRNA varies during the cycle, with maximum leveis in predivisional cells. Western blot analysis of GroEL leveis in Caulobaaer has shown that the amount of the protein decreases during the first 60 minutes of C.crescentus cell cycle and then starts to increase again. These results corroborate the data obtained with Northern blot analysis. A similar experiment was performed after exposing a mixed population of C.crescentus cells to different times of heat shock at 400C. Western blot of extracts of these cells showed a fivefold increase in the leveis of GroEL after 60 minutes of heat shock, which then begins to decrease. Primer extension experiments were performed using total RNA from cells incubated at normal growth temperature or after heat shock treatment. Two possible transcription start sites were determined at positions -119 and -88 from the ATG of the groES initiator methionine and the -10 and -35 regions of the corresponding promoters (P 1 and P2) were identified. Only trancription initiating from the P2 promoter, which has caracteristics of a σ32 promoter, I ncreases during heat shock .Transcription fusions with the reporter vector placZ/290 and the 5\' regulatory region of the groESL operan were contructed in order to identify the sequences responsible for heat shock and cell cycle contral. Deletion analysis in the 5\' region of the operon showed that no sequences upstream of the P2 promoter are necessary for heat shock induction or for temporal contral. Site-directed mutagenesis in the inverted repeat found 3\' of the P2 promoter, in front of the groES gene, revealed that this element, also known as CIRCE, negatively regulates groESL expression at 300C and mutations in it lead to loss of temporal control of this heat shock operon.

Made available in DSpace on 2014-06-11T19:23:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-11-16Bitstream added on 2014-06-13T19:28:59Z : No. of bitstreams: 1 ucci_ap_me_araiq.pdf: 1107212 bytes, checksum: d5908c197d6d3cb915d0549cd1755e02 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Xanthomonas citri subsp. citri (Xcc) é uma bactéria Gram-negativa responsável pelo o cancro cítrico. A doença causada por este fitopatógeno constitui numa das mais graves doenças da citricultura brasileira, cujas medidas de controle capazes de eliminar completamente a doença são ineficientes até o momento. O sequenciamento e anotação do genoma de Xcc juntamente com estudos relacionados à genômica funcional nesta bactéria realizados em nosso laboratório mostraram a existência, na bactéria, de alguns genes (copA e copB) que codificam proteínas envolvidas com o mecanismo de resistência a cobre (CopA e CopB). Os genes que codificam ambas proteínas estão organizados em um operon, cuja transcrição é induzida por cobre e é específica ao metal. Componentes a base de cobre são comumente usados na agricultura como agentes microbicidas a fim de reduzir a severidade desta e também de outras doenças nas plantas. Contudo, a eficácia do cobre está sendo reduzida pelo aparecimento de linhagens de bactérias resistentes a este metal, portanto, estudos visando a compreensão dos mecanismos moleculares envolvidos na resistência a cobre em Xcc são de grande importância. Uma pequena ORF (XAC3629) que codifica uma proteína hipotética de 152 aminoácidos e que se localiza upstream aos genes copAB foi identificada. Neste trabalho foi estudado o papel da ORF na regulação do operon copAB. Linhagens mutantes contendo a ORF XAC3629 interrompida por um transposon (XAC3629 / Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium that causes citrus canker, one of the most serious diseases in Brazilian crops, and the ways to eliminate it are not efficient. After sequencing and annotation of the Xcc genome our laboratory started to perform functional genomic studies in Xcc. Previous studies identified two genes (copA and copB) that encode proteins involved in the copper resistance mechanism (CopA and CopB). The genes are organized in an operon whose transcription is induced and specific to copper. Copper compounds are commonly used in agriculture in order to control plant diseases, however the copper effectiveness is being reduced due to the emergence of copper resistant bacteria strains. Therefore, studies related to copper resistance mechanism in Xcc are very relevant. A short ORF (XAC3629) which encodes an 152 amino acids hypothetical protein was identified upstream the operon copAB. In this work the role of the ORF in the operon regulation was studied. Mutant strains containing the ORF XAC3629 interrupetd by a transposon (XAC3629

Orientador: Maria Célia Bertolini / Banca: Jesus Aparecido Ferro / Banca: Julio Cezar Franco de Oliveira / Resumo: Xanthomonas citri subsp. citri (Xcc) é uma bactéria Gram-negativa responsável pelo o cancro cítrico. A doença causada por este fitopatógeno constitui numa das mais graves doenças da citricultura brasileira, cujas medidas de controle capazes de eliminar completamente a doença são ineficientes até o momento. O sequenciamento e anotação do genoma de Xcc juntamente com estudos relacionados à genômica funcional nesta bactéria realizados em nosso laboratório mostraram a existência, na bactéria, de alguns genes (copA e copB) que codificam proteínas envolvidas com o mecanismo de resistência a cobre (CopA e CopB). Os genes que codificam ambas proteínas estão organizados em um operon, cuja transcrição é induzida por cobre e é específica ao metal. Componentes a base de cobre são comumente usados na agricultura como agentes microbicidas a fim de reduzir a severidade desta e também de outras doenças nas plantas. Contudo, a eficácia do cobre está sendo reduzida pelo aparecimento de linhagens de bactérias resistentes a este metal, portanto, estudos visando a compreensão dos mecanismos moleculares envolvidos na resistência a cobre em Xcc são de grande importância. Uma pequena ORF (XAC3629) que codifica uma proteína hipotética de 152 aminoácidos e que se localiza upstream aos genes copAB foi identificada. Neste trabalho foi estudado o papel da ORF na regulação do operon copAB. Linhagens mutantes contendo a ORF XAC3629 interrompida por um transposon (XAC3629::Tn), previamente construídas no laboratório, foram avaliadas em relação ao crescimento em meio contendo diferentes concentrações de cobre. Estas linhagens mostraram perda da resistência quando o metal foi adicionado ao meio, mesmo em baixas concentrações (0,25 mM), sugerindo seu envolvimento na regulação do operon de resistência a cobre copAB. Células da linhagem selvagem ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium that causes citrus canker, one of the most serious diseases in Brazilian crops, and the ways to eliminate it are not efficient. After sequencing and annotation of the Xcc genome our laboratory started to perform functional genomic studies in Xcc. Previous studies identified two genes (copA and copB) that encode proteins involved in the copper resistance mechanism (CopA and CopB). The genes are organized in an operon whose transcription is induced and specific to copper. Copper compounds are commonly used in agriculture in order to control plant diseases, however the copper effectiveness is being reduced due to the emergence of copper resistant bacteria strains. Therefore, studies related to copper resistance mechanism in Xcc are very relevant. A short ORF (XAC3629) which encodes an 152 amino acids hypothetical protein was identified upstream the operon copAB. In this work the role of the ORF in the operon regulation was studied. Mutant strains containing the ORF XAC3629 interrupetd by a transposon (XAC3629::Tn) were previously constructed in our laboratory and in this work their growth were evaluated in a medium containing different copper concentration. The mutant strains were unable to grow when the metal was added to the medium, even at low concentration (0.25 mM) suggesting that the ORF XAC3629 may have a role in regulating the operon copAB. Wild type Xcc cells are able to grow up to 1 mM copper concentration. Attempts to construct an ORF XAC3629 knocked out strain were performed but this mutant strain was not obtained. Two XAC3629::Tn mutants strains were transformed with a plasmid construction containing the ORF XAC3629 entire sequence plus its flanking regions. The results did not show functional complementation (recovery of the copper resistance when cells were grown in medium containing ... (Complete abstract click electronic access below) / Mestre

The idea of morphogen gradients encoding positional information for a developing organism has long been discussed in the field of developmental biology, but only recently have quantitative models been proposed that relate measured transcription factor concentrations to enhancer activity. However, successful models are typically computationally time-consuming, thus limiting full exploration and interpretation of the data. This thesis addresses these problems using standard statistical techniques applied to a comprehensive data set with the even skipped (eve) locus as a test case. The first part of the thesis introduces the data set. This is the precellular Virtual Embryo from the Berkeley Drosophila Transcription Network project. It comprises expression measurements of almost 100 genes in more than 6,000 individual nuclei at six time points. Different modelling approaches are evaluated in the context of this data set leading to a justification of logistic regression and the methods used to prepare the data set for further analysis. The second part applies logistic regression to describe the response of the eve enhancers to known regulating transcription factors such as Hunchback. Predictions of behaviour under regulator perturbation are consistent with experimental results and the functional form is shown not to be arbitrarily flexible, both in terms of the regulators and regions of the embryo included. The third part uses the framework developed above to find minimal explanatory models in the context of statistical model selection. It is found that the best scoring models depend on well-known regulators. The model selection techniques are then extended by directing the process using previous biological observations to analyse the eve 2 and eve 3+7 enhancers. The results are consistent with published research, but suggest specific additional hypotheses for the enhancers' regulation. Finally, the thesis concludes by proposing a general model of positional information and discussing the biological implications of the results. Overall, the results show how transcriptional control can be allocated to discrete enhancers and that characterising their activity in relatively simple terms is sufficient to explain their precise spatially-defined response to transcription factor concentrations.

One of the greatest challenges in modern biology is to understand quantitatively the mechanisms underlying messenger Ribonucleic acid (mRNA) transcription within the cell. To this end, integrated functional genomics attempts to use the vast wealth of data produced by modern large scale genomic projects to understand how the genome is deployed to create a diversity of tissues and species. The expression levels of tens or hundreds of thousands genes are profiled at multiple time points or different experimental conditions in the genomic projects. The profiling results are deposited in large scale quantitative data files that are not possible to analyse without systematic computational methods. In particular, it is much more difficult to experimentally measure the concentration level of transcription factor proteins and their affinity for the promoter region of genes, while it is relatively easy to measure the result of transcription using experimental techniques such as microarrays. In the absence of such biological experiments, it becomes necessary to use in silico techniques to determine the transcription factor regulatory activities given existing gene expression profile data. It therefore presents significant challenges and opportunities to the computer science community. This PhD Project made use of one such in silico technique to determine the differences (if any) in transcription factor regulatory activities of different experimental conditions and time points.The research aim of the Project was to understand the transcriptional regulatory mechanism that controls the sophisticated process of gene expression in cells. In particular, differences in the downstream signalling from which transcription factors can play a role in predisposition to diseases such as Parasitic disease, Cancer, and Neuroendocrine disease. To address this question I have had access to large integrated genomics datasets generated in studies on parasitic disease, lung cancer, and endocrine (hormone) disease. The current state-of-the-art takes existing knowledge and asks "How do these data relate to what we already know?" By applying machine learning approaches the project explored the role that such data can play in uncovering new biological knowledge.

The past two decades have highlighted the utility in using the fruit fly Drosophila as a model organism for unravelling the molecular and functional complexities of two important fields of research: the systems that guide retinal determination (RD) and circadian rhythms (the daily body clock or oscillator). RD and clock factors are of great interest as they are: (1) highly conserved in vertebrates; (2) also essential for other physiological systems; (3) implicated in several congenital disorders and other diseases. The RD factors operate within a network in which several of their interactions have been described. Two such factors, eyes absent (eya) and sine oculis (so), are known to function as a unit to direct transcriptional regulation during photoreceptor (PR) differentiation. The regulation of eya and so by a transcriptional repressor at the heart of the clock mechanism, vrille (vri), is here investigated. Two distinct observations advocated exploration of a link between vri and eya/ so is of interest. (1) vri is a core component of the clock and interacts with RD but the RD function is unknown. (2) Recent evidence suggests that an RD factor directly upstream of eya and so, twin-of-eyeless (toy), interacts with the oscillator mechanism through direct and indirect pathways. It is possible that the indirect influences of toy on the oscillator are mediated via eya and/ or so. Interactions between eya, so and vri during RD and within oscillator cells are investigated here.Eye development function was studied using immunohistochemistry and transgenic manipulation. VRI is not expressed within the developed PRs; rather, expression of VRI is down-regulated prior to differentiation. In addition, conversely to the hypothesised role, VRI is co-expressed with EYA in some regions. Together with data from transgenic manipulation of VRI regional expression, I propose that VRI is predominantly part of a developmental pathway but can attenuate eya and so expression.The VRI binding site has been described previously and several sites were identified within eya/ so loci, some of which were tested in an in vitro binding assay. Two such sites were located adjacent to a known enhancer of so. I generated two transgenic fly lines containing: 1) an extension of the original enhancer to contain the VRI sites; and 2) a similar construct with the VRI sites ablated. Comparison of the original enhancer to those from the current study confirmed that the VRI sites attenuate expression and that intervening regions must contain binding sites for other transcription factors.In adult brains over a circadian light-dark cycle, EYA protein was expressed in three of the central brain clock neurones. Furthermore, expression of eya and so transcripts in adult heads, PRs and the brain, changed over the light/ dark cycle independently of the clock - indicating that their expression is modulated over the light-dark cycle but not by the oscillator mechanism. These data suggest interactions between eye development factors eya/ so and oscillator components, or, the light/ dark cycle exist. These interactions may be important for tissue-specific circadian physiology as well as the overall oscillator mechanism and offer an intriguing route for future investigation.

The nuclear lamina is a filamentous network that underlies the nuclear envelope. Lamina components include the family of LEM domain (LEM-D) proteins, named for LAP2, emerin and MAN1. Mutations in genes encoding LEM-D proteins cause tissue-restricted human disease, even though these genes are globally expressed. To understand the contributions of the LEM-D proteins to nuclear lamina function, investigations of the Drosophila LEM-D proteins was undertaken. The Drosophila genome encodes four LEM-D proteins and this thesis describes work done on the Drosophila homologues of MAN1 and emerin, Drosophila MAN1 (dMAN1) and Otefin (Ote). Chapter 2 describes the generation and phenotypic analyses of dMAN1 mutants. These mutants display a range of tissue-specific defects associated with an increase in BMP/Dpp signaling. This suggests that dMAN1 downregulates BMP/Dpp signaling at the nuclear periphery. Chapter 3 describes the identification and phenotypic analyses of ote mutants. Loss of Ote is associated with a tissue-specific defect of the female germline where ote mutant females display defects in germline stem cell (GSC) maintenance. Loss of Ote causes defects in the germline cells, the cap cells of GSC niche and an increased sensitivity to Dpp signaling in both germline and somatic cells. These findings support models suggesting that laminopathies arise from dysfunction of the homeostasis in stem cell populations. Taken together, these studies suggest that the nuclear lamina may play tissue-specific roles through regulation of signal transduction pathways. Our data also support the use of Drosophila as a system to elucidate the mechanistic basis of diseases associated with defects in the nuclear lamina.

No description available.

The goal of the ENCODE project has been to characterize regulatory elements in the human genome, such as regions bound by transcription factors (TFs), regions of open chromatin and regions with altered histone modifications. The ENCODE consortium has performed a large number of whole-genome experiments to measure TF binding, chromatin accessibility, gene expression and histone modifications, on a multitude of cell types and conditions in both human and mouse. In this dissertation I describe the analysis of numerous datasets comprising 66 epigenomes, chromatin accessibility and expression data across twelve tissues and seven time points, during mouse embryonic development. We defined chromatin states using histone modification data and performed integrative analysis on the states. We observed coordinated changes of histone mark signals at enhancers and promoters with gene expression. We detected evolutionary conserved bivalent promoters, selectively silencing ~3,400 genes, including hundreds of TFs regulating embryonic development. Second, I present a supervised method to predict TF binding across cell types, with features based on DNA sequence and patterns in DNase I cleavage data. We found that sequence and DNase read counts can outperform other features as well as state-of-the-art methods. I also describe our contribution to the ENCODE TF Binding DREAM challenge where we developed a method, using multiscale features and Extreme Boosting. Third, I describe methods, tools, and computational infrastructure that we have developed to handle large amounts of experimental data and metadata. These tools are fundamental to the selection and integration of large experimental datasets and are at the core of our pipelines, which are described in this dissertation. Finally, I present the protein docking server I developed, as well as algorithms and routines for post-processing predictions and protein structures. Collectively, this body of work encompasses computational approaches to the analyses of chromatin states, gene regulation, and the integration of large experimental datasets. / 2021-08-31T00:00:00Z

Pluripotent stem cells have the capacity to develop into different cell lineages and can be manipulated into certain cell types through the use of small molecule inducers. Retinoic acid (RA) signaling through retinoic acid receptors (RAR) and retinoid X receptors (RXR) has the ability to direct lineage determination but has yielded disappointing results in promoting skeletal myogenesis in embryonic stem (ES) cells. RXR is crucial in embryonic development although it is generally considered to act as a silent partner for other nuclear receptors such as RAR. Our findings demonstrate that rexinoid specific signaling enhances skeletal myogenesis and requires β-catenin but not RAR. Moreover, RXR signalling in mouse ES cells can efficiently enhance skeletal myogenesis and closely recapitulates sequential events observed in vivo. Since ES cells closely represent the properties of the developing embryo, efficiently generating skeletal muscle provides a means to further scrutinize signaling pathways in myogenic development in view of developing therapies for muscle related diseases.