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Caracterização dos fatores sigma da RNA polimerase do fitopatógeno Xanthononas axonopodis pv. citri / Caracterization of RNA polimerase sigma factor of phythopathogen Xanthomonas axonopodis pv. citri.Francischini, Maria Claudia Pereda 01 October 2010 (has links)
A citricultura é de grande importância para as atividades agrícolas brasileiras, uma vez que o Brasil é o principal produtor e exportador de suco de laranja. O cancro cítrico, causado pela bactéria Xanthomonas axonopodis pv. citri (Xac) é um grave problema nesse setor, causando um elevado prejuízo na produção de frutos e seus derivados. O fator sigma é a subunidade da RNA polimerase que tem a função de direcionar o núcleo da RNA polimerase a uma classe específica de sequências promotoras. Como a maioria das bactérias sintetiza diversos fatores sigma, essa característica proporciona à bactéria a oportunidade de manutenção basal da sua expressão gênica, assim como, a regulação em resposta a alterações ambientais e a sinais durante o desenvolvimento bacteriano. O genoma de Xac codifica para 14 fatores sigma. Nesse presente trabalho, detectamos interações dos fatores σECF (Xac2814. Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) e seus fatores anti-σ cognatos (Xac2815. Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). Além disso, observamos interações entre o fator σFliA (Xac1933) e o anti-σFlgM (Xac1989), seu fator anti-σ cognato. A caracterização das cepas nocautes para alguns fatores σ apontaram o envolvimento do fator σ54Xac1969 no mecanismo de formação de flagelo, a contribuição do fator σECFXac1682 na resposta ao choque térmico e a participação do fator σECFXac2191 no crescimento bacteriano em condições de carência de ferro. / Citriculture is an important sector of the economy of the State of São Paulo. Citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac), is a devastating disease responsible for large agribusiness losses every year. several sigma factors. The sigma factor is the subunit of RNA polymerase that serves to direct the RNA polymerase core to a specific class of promoter sequences. Most bacteria code for more than one sigma factor, which provides the cell with the means by which to maintain basal gene expression while at the same time modulate the expression of specific genes in response in environmental changes and signals during bacterial growth. The Xac genome codes for 14 sigma factors which are the objects of study in this thesis. We demonstrate that many of the sigma factors of the σECF family (Xac2814, Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) interact with cognate anti- factors (Xac2815, Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). These sigma-anti-sigma pairs are all coded by neighboring genes. Interactions between the sigma factor σFliA (Xac1933) and anti-σFlgM (Xac1989) were also observed. Xac strains with gene knockouts for several sigma factors were produced. The characterization some these knockout strains point to the involvement of σ54Xac1969 in the biosynthesis of flagella, participation of σECFXac1682 in the ability to survive heat shock and involvement of σECFXac2191 in the response to iron deficiency.
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Probing sequence-level instructions for gene expression / Etude des instructions pour l’expression des gènes présentes dans la séquence ADNTaha, May 28 November 2018 (has links)
La régulation des gènes est fortement contrôlée afin d’assurer une large variété de types cellulaires ayant des fonctions spécifiques. Ces contrôles prennent place à différents niveaux et sont associés à différentes régions génomiques régulatrices. Il est donc essentiel de comprendre les mécanismes à la base des régulations géniques dans les différents types cellulaires, dans le but d’identifier les régulateurs clés. Plusieurs études tentent de mieux comprendre les mécanismes de régulation en modulant l’expression des gènes par des approches épigénétiques. Cependant, ces approches sont basées sur des données expérimentales limitées à quelques échantillons, et sont à la fois couteuses et chronophages. Par ailleurs, les constituants nécessaires à la régulation des gènes au niveau des séquences ne peut pas être capturées par ces approches. L’objectif principal de cette thèse est d’expliquer l’expression des ARNm en se basant uniquement sur les séquences d’ADN.Dans une première partie, nous utilisons le modèle de régression linéaire avec pénalisation Lasso pour prédire l’expression des gènes par l’intermédiaire des caractéristique de l’ADN comme la composition nucléotidique et les sites de fixation des facteurs de transcription. La précision de cette approche a été mesurée sur plusieurs données provenant de la base de donnée TCGA et nous avons trouvé des performances similaires aux modèles ajustés aux données expérimentales. Nous avons montré que la composition nucléotidique a un impact majeur sur l’expression des gènes. De plus, l’influence de chaque régions régulatrices est évaluée et l’effet du corps de gène, spécialement les introns semble être clé dans la prédiction de l’expression. En second partie, nous présentons une tentative d’amélioration des performances du modèle. D’abord, nous considérons inclure dans le modèles les interactions entres les différents variables et appliquer des transformations non linéaires sur les variables prédictives. Cela induit une légère augmentation des performances du modèles. Pour aller plus loin, des modèles d’apprentissage profond sont étudiés. Deux types de réseaux de neurones sont considérés : Les perceptrons multicouches et les réseaux de convolutions.Les paramètres de chaque neurone sont optimisés. Les performances des deux types de réseaux semblent être plus élevées que celles du modèle de régression linéaire pénalisée par Lasso. Les travaux de cette thèse nous ont permis (i) de démontrer l’existence des instructions au niveau de la séquence en relation avec l’expression des gènes, et (ii) de fournir différents cadres de travail basés sur des approches complémentaires. Des travaux complémentaires sont en cours en particulier sur le deep learning, dans le but de détecter des informations supplémentaires présentes dans les séquences. / Gene regulation is tightly controlled to ensure a wide variety of cell types and functions. These controls take place at different levels and are associated with different genomic regulatory regions. An actual challenge is to understand how the gene regulation machinery works in each cell type and to identify the most important regulators. Several studies attempt to understand the regulatory mechanisms by modeling gene expression using epigenetic marks. Nonetheless, these approaches rely on experimental data which are limited to some samples, costly and time-consuming. Besides, the important component of gene regulation based at the sequence level cannot be captured by these approaches. The main objective of this thesis is to explain mRNA expression based only on DNA sequences features. In a first work, we use Lasso penalized linear regression to predict gene expression using DNA features such as transcription factor binding site (motifs) and nucleotide compositions. We measured the accuracy of our approach on several data from the TCGA database and find similar performance as that of models fitted with experimental data. In addition, we show that nucleotide compositions of different regulatory regions have a major impact on gene expression. Furthermore, we rank the influence of each regulatory regions and show a strong effect of the gene body, especially introns.In a second part, we try to increase the performances of the model. We first consider adding interactions between nucleotide compositions and applying non-linear transformations on predictive variables. This induces a slight increase in model performances.To go one step further, we then learn deep neuronal networks. We consider two types of neural networks: multilayer perceptrons and convolution networks. Hyperparameters of each network are optimized. The performances of both types of networks appear slightly higher than those of a Lasso penalized linear model. In this thesis, we were able to (i) demonstrate the existence of sequence-level instructions for gene expression and (ii) provide different frameworks based on complementary approaches. Additional work is ongoing, in particular with the last direction based on deep learning, with the aim of detecting additional information present in the sequence.
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Análise do metabolismo de polifosfato e do operon pst em Pseudomonas aeruginosa. / Analysis of the metabolism of polyphosphate and of the pst operon in Pseudomonas aeruginosa.Munevar, Nicolas Federico Villamil 06 August 2015 (has links)
O operon pst de P. aeruginosa codifica um transportador de fosfato de alta afinida-de e também a proteína PhoU que, em conjunto, atuam como repressores da ex-pressão do regulon Pho dessa espécie. A atividade de PhoU está também associada ao metabolismo de polifosfato (poliP), dado que mutantes phoU nulos apresentam um vasto acúmulo do biopolímero. Ensaios de β-galactosidase mostraram uma alteração na expressão dos genes ppk e ppx, envolvidos no metabolismo de poliP, no mutante phoU. Observou-se que na cepa selvagem, a transcrição de ppk e de ppx não responde às limitações de Pi ou de nitrogênio, sendo esses genes altamente expressos em condições normais de crescimento. Além disso, determinou-se que ppk é co-transcrito com o gene hemB, os quais formam, portanto, um operon. O operon pst também foi analisado. Foi identificado por ensaios de northern blot o transcrito do primeiro gene do operon, pstS, que codifica uma proteína periplasmática. Também, foi identificado um promotor imediatamente a montante de phoU, o gene mais distal do operon, que permitiria sua expressão em condições normais do crescimento bacteriano. Por fim, determinou-se por ensaios de EMSA que as duas sequências consenso Pho box presentes no operon pst são completamente funcionais. / The pst operon in P. aeruginosa encodes a high-affinity phosphate transporter and the PhoU protein, which together act as repressors of Pho regulon of this species. The PhoU activity is also related with polyphosphate (polyP) metabolism, since phoU null mutants have a large accumulation of the biopolymer. β-galactosidase assays allowed to confirm a change in the expression of ppk and ppx genes, in-volved in PolyP metabolism, in the phoU mutant. It was also evidenced that in the wild type strain, the ppk and ppx transcription does not respond to Pi or nitrogen starvation, and that these genes are highly expressed under conditions of normal growth. In addition, it was determined that ppk is co-transcribed with hemB, a gene involved in the synthesis of porphyrins, and they constitute therefore an operon. The pst operon was also examined. Was identified by northern blot the transcript of the first gene in the operon, pstS, which encodes a periplasmic protein. Also, a promoter was identified immediately upstream of phoU, the most distal gene in the operon, allowing its expression in normal conditions of bacterial growth. Finally, it was determined by EMSA that the two consensus sequences Pho box present in the pst operon are fully functional.
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TARP Promoter-Based Prostate Cancer Gene Therapy : From Development to ApplicationCheng, Wing-Shing January 2005 (has links)
<p>Prostate cancer is one leading cause of cancer-related death among men in Western countries. The standard therapies for localized prostate cancer include radical prostatectomy and radiation therapy. Such measures are relatively effective in the short term, but many patients ultimately relapse. These patients may benefit from a combination of standard therapy and oncolytic virus therapy. My work aimed to develop viruses for this purpose.</p><p>TARP is a protein that in males is specifically expressed in prostate epithelial and cancer cells. In my thesis, I characterized the TARP promoter and showed that TARP expression is regulated at the transcriptional level by testosterone through binding of the androgen receptor in the proximal TARP promoter. I further developed TARP promoter-based regulatory sequences for prostate-specific gene expression. A sequence comprising a PSA enhancer, a PSMA enhancer and the TARP promoter was constructed and designated PPT. An adenoviral vector containing the PPT sequence shielded from transcriptional interference by an H19 insulator showed high prostate-specific transcriptional activity in human cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer the PPT sequence is not active. Therefore, a two-step transcriptional amplification (TSTA) system was used together with the PPT sequence to develop an adenovirus that confers prostate-specific transgene expression also in murine cells.</p><p>I constructed a conditionally replicating adenovirus where the E1A gene expression is controlled by an H19 insulator-shielded PPT regulatory sequence, Ad[I/PPT-E1A]. This virus exhibited absolute prostate specificity in terms of E1A expression, viral replication and cytolysis <i>in vitro</i> and <i>in vivo</i>. Importantly, our virus is active both in the presence and absence of testosterone, which may prove beneficial for patients treated by hormonal withdrawal. </p><p>Hopefully, my work will improve existing gene therapy strategies for prostate cancer and in the long term improve the prognosis for patients with prostate cancer.</p>
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Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase GeneSöderberg, Malin January 2005 (has links)
<p>Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented.</p><p>Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA.</p><p>Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. <i>Trans</i>-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs.</p><p>In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.</p>
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Theory of mRNA degradationDeneke, Carlus January 2012 (has links)
One of the central themes of biology is to understand how individual cells achieve a high fidelity in gene expression. Each cell needs to ensure accurate protein levels for its proper functioning and its capability to proliferate. Therefore, complex regulatory mechanisms have evolved in order to render the expression of each gene dependent on the expression level of (all) other genes. Regulation can occur at different stages within the framework of the central dogma of molecular biology. One very effective and relatively direct mechanism concerns the regulation of the stability of mRNAs. All organisms have evolved diverse and powerful mechanisms to achieve this. In order to better comprehend the regulation in living cells, biochemists have studied specific degradation mechanisms in detail. In addition to that, modern high-throughput techniques allow to obtain quantitative data on a global scale by parallel analysis of the decay patterns of many different mRNAs from different genes.
In previous studies, the interpretation of these mRNA decay experiments relied on a simple theoretical description based on an exponential decay. However, this does not account for the complexity of the responsible mechanisms and, as a consequence, the exponential decay is often not in agreement with the experimental decay patterns.
We have developed an improved and more general theory of mRNA degradation which provides a general framework of mRNA expression and allows describing specific degradation mechanisms. We have made an attempt to provide detailed models for the regulation in different organisms. In the yeast S. cerevisiae, different degradation pathways are known to compete and furthermore most of them rely on the biochemical modification of mRNA molecules. In bacteria such as E. coli, degradation proceeds primarily endonucleolytically, i.e. it is governed by the initial cleavage within the coding region. In addition, it is often coupled to the level of maturity and the size of the polysome of an mRNA. Both for S. cerevisiae and E. coli, our descriptions lead to a considerable improvement of the interpretation of experimental data. The general outcome is that the degradation of mRNA must be described by an age-dependent degradation rate, which can be interpreted as a consequence of molecular aging of mRNAs. Within our theory, we find adequate ways to address this much debated topic from a theoretical perspective.
The improvements of the understanding of mRNA degradation can be readily applied to further comprehend the mRNA expression under different internal or environmental conditions such as after the induction of transcription or stress application. Also, the role of mRNA decay can be assessed in the context of translation and protein synthesis.
The ultimate goal in understanding gene regulation mediated by mRNA stability will be to identify the relevance and biological function of different mechanisms. Once more quantitative data will become available, our description allows to elaborate the role of each mechanism by devising a suitable model. / Ein zentrales Ziel der modernen Biologie ist es, ein umfassendes Verständnis der Genexpression zu erlangen. Die fundamentalen Prozesse sind im zentralen Dogma der Genexpression zusammengefasst: Die genetische Information wird von DNA in Boten-RNAs (mRNA) transkribiert und im Prozess der Translation von mRNA in Proteine übersetzt. Zum Erhalt ihrer Funktionalität und der Möglichkeit von Wachstum und Fortpflanzung muss in jeder Zelle und für jedes Gen die optimale Proteinkonzentration akkurat eingestellt werden. Hierzu hat jeder Organismus detaillierte Regulationsmechanismen entwickelt. Regulation kann auf allen Stufen der Genexpression erfolgen, insbesondere liefert der Abbau der mRNA-Moleküle einen effizienten und direkten Kontrollmechanismus. Daher sind in allen Lebewesen spezifische Mechanismen - die Degradationsmechanismen - entstanden, welche aktiv den Abbau befördern. Um ein besseres Verständnis von den zugrunde liegenden Prozessen zu erlangen, untersuchen Biochemiker die Degradationsmechanismen im Detail. Gleichzeitig erlauben moderne molekularbiologische Verfahren die simultane Bestimmung der Zerfallskurven von mRNA für alle untersuchten Gene einer Zelle. Aus theoretischer Perspektive wird der Zerfall der mRNA-Menge als exponentieller Zerfall mit konstanter Rate betrachtet. Diese Betrachtung dient der Interpretation der zugrunde liegenden Experimente, berücksichtigt aber nicht die fundierten Kenntnisse über die molekularen Mechanismen der Degradation. Zudem zeigen viele experimentelle Studien ein deutliches Abweichen von einem exponentiellen Zerfall.
In der vorliegenden Doktorarbeit wird daher eine erweiterte theoretische Beschreibung für die Expression von mRNA-Molekülen eingeführt. Insbesondere lag der Schwerpunkt auf einer verbesserten Beschreibung des Prozesses der Degradation. Die Genexpression kann als ein stochastischer Prozess aufgefasst werden, in dem alle Einzelprozesse auf zufällig ablaufenden chemischen Reaktionen basieren. Die Beschreibung erfolgt daher im Rahmen von Methoden der stochastischen Modellierung. Die fundamentale Annahme besteht darin, dass jedes mRNA-Molekül eine zufällige Lebenszeit hat und diese Lebenszeit für jedes Gen durch eine statistische Lebenszeitverteilung gegeben ist. Ziel ist es nun, spezifische Lebenszeitverteilungen basierend auf den molekularen Degradationsmechanismen zu finden. In dieser Arbeit wurden theoretische Modelle für die Degradation in zwei verschiedenen Organismen entwickelt.
Zum einen ist bekannt, dass in eukaryotischen Zellen wie dem Hefepilz S. cerevisiae mehrere Mechanismen zum Abbau der mRNA-Moleküle in Konkurrenz zueinander stehen. Zudem ist der Abbau durch mehrere geschwindigkeitsbestimmende biochemische Schritte charakterisiert. In der vorliegenden Arbeit wurden diese Feststellungen durch ein theoretisches Modell beschrieben. Eine Markow-Kette stellte sich als sehr erfolgreich heraus, um diese Komplexität in eine mathematisch-fassbare Form abzubilden.
Zum anderen wird in Kolibakterien die Degradation überwiegend durch einen initialen Schnitt in der kodierenden Sequenz der mRNA eingeleitet. Des Weiteren gibt es komplexe Wechselwirkungen mit dem Prozess der Translation. Die dafür verantwortlichen Enzyme - die Ribosomen - schützen Teile der mRNA und vermindern dadurch deren Zerfall. In der vorliegenden Arbeit wurden diese Zusammenhänge im Rahmen eines weiteren spezifischen, theoretischen Modells untersucht.
Beide Mechanismen konnten an experimentellen Daten verifiziert werden. Unter anderem konnten dadurch die Interpretation der Zerfallsexperimente deutlich verbessert und fundamentale Eigenschaften der mRNA-Moleküle bestimmt werden.
Ein Vorteil der statistischen Herangehensweise in dieser Arbeit liegt darin, dass theoretische Konzepte für das molekulare Altern der mRNAs entwickelt werden konnten. Mit Hilfe dieser neuentwickelten Methode konnte gezeigt werden, dass sich die Komplexität der Abbaumechanismen in einem Alterungsprozess manifestiert. Dieser kann mit der Lebenserwartung von einzelnen mRNA-Molekülen beschrieben werden.
In dieser Doktorarbeit wurde eine verallgemeinerte theoretische Beschreibung des Abbaus von mRNAMolek ülen entwickelt. Die zentrale Idee basiert auf der Verknüpfung von experimentellen Zerfallsmessungen mit den biochemischen Mechanismen der Degradation. In zukünftigen experimentellen Untersuchungen können die entwickelten Verfahren angewandt werden, um eine genauere Interpretation der Befunde zu ermöglichen. Insbesondere zeigt die Arbeit auf, wie verschiedene Hypothesen über den Degradationsmechanismus anhand eines geeigneten mathematischen Modells durch quantitative Experimente verifiziert oder falsifiziert werden können.
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TARP Promoter-Based Prostate Cancer Gene Therapy : From Development to ApplicationCheng, Wing-Shing January 2005 (has links)
Prostate cancer is one leading cause of cancer-related death among men in Western countries. The standard therapies for localized prostate cancer include radical prostatectomy and radiation therapy. Such measures are relatively effective in the short term, but many patients ultimately relapse. These patients may benefit from a combination of standard therapy and oncolytic virus therapy. My work aimed to develop viruses for this purpose. TARP is a protein that in males is specifically expressed in prostate epithelial and cancer cells. In my thesis, I characterized the TARP promoter and showed that TARP expression is regulated at the transcriptional level by testosterone through binding of the androgen receptor in the proximal TARP promoter. I further developed TARP promoter-based regulatory sequences for prostate-specific gene expression. A sequence comprising a PSA enhancer, a PSMA enhancer and the TARP promoter was constructed and designated PPT. An adenoviral vector containing the PPT sequence shielded from transcriptional interference by an H19 insulator showed high prostate-specific transcriptional activity in human cells both in the presence and absence of testosterone. However, in experimental murine prostate cancer the PPT sequence is not active. Therefore, a two-step transcriptional amplification (TSTA) system was used together with the PPT sequence to develop an adenovirus that confers prostate-specific transgene expression also in murine cells. I constructed a conditionally replicating adenovirus where the E1A gene expression is controlled by an H19 insulator-shielded PPT regulatory sequence, Ad[I/PPT-E1A]. This virus exhibited absolute prostate specificity in terms of E1A expression, viral replication and cytolysis in vitro and in vivo. Importantly, our virus is active both in the presence and absence of testosterone, which may prove beneficial for patients treated by hormonal withdrawal. Hopefully, my work will improve existing gene therapy strategies for prostate cancer and in the long term improve the prognosis for patients with prostate cancer.
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Post-Transcriptional Regulation of the Murine Inducible Nitric Oxide Synthase GeneSöderberg, Malin January 2005 (has links)
Large amounts of nitric oxide (NO) are produced by the inducible nitric oxide synthase (iNOS) upon inflammatory stimuli. NO is a multifaceted molecule, which may have beneficial effects as an antimicrobial agent in the immune defense, or cytotoxic effects in chronic inflammations, manifested as e.g. arthritis and asthma. Understanding the mode of regulation of the iNOS gene is a prerequisite for developing intervention strategies in various pathological conditions where detrimental effects of NO need to be prevented. Transcriptional processes of the iNOS gene regulation are well described, while post-transcriptional events have not been studied in detail. The aim of the present thesis was to investigate post-transcriptional regulatory mechanisms involving the 3’untranslated region (UTR) of the murine iNOS mRNA. Inflammation-dependent RNA-protein interactions with the iNOS mRNA 3’UTR were characterized by RNA gel shift analysis and UV cross-linking. Trans-acting factors interacting with the 3’UTR were detected in mouse liver and macrophages and identified as heterogeneous nuclear ribonucleoproteins (hnRNP) I and L. Western blot revealed that reduced hnRNPI levels are responsible for the decreased interaction of hnRNPI with iNOS 3’UTR upon induction in inflammatory conditions. This decrease was reversed by the glucocorticoid dexamethasone, concomitant with decreased iNOS mRNA levels and stability. Introduction of the iNOS 3’UTR into a luciferase reporter gene reduced its expression in macrophages. Upon deletions of the binding sites for hnRNPI and hnRNPL, the luciferase expression was recovered. In addition, inflammatory stimuli increased the luciferase activity of the construct with the full-length 3’UTR, while only weak effects of the stimuli were seen on the deletion constructs. In conclusion, the results suggest that binding of hnRNPI and hnRNPL to the iNOS mRNA 3’UTR promotes degradation of the transcript. Induction of iNOS by inflammatory stimuli dissociates the RNA-protein complex, yielding a more stable mRNA. In addition, post-transcriptional down-regulation of the iNOS gene by the anti-inflammatory glucocorticoid dexamethasone, seems to involve hnRNPI.
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Newer Insights On Structure, Function And Regulation Of Dps Protein From Mycobacterium smegmatisChowdhury, Rakhi Pait 06 1900 (has links)
The first chapter will provide an introduction to the physiology, pathogenesis and biology of mycobacteria. Host-pathogen interactions, different modes of resistance of the bacteria, adaptations for survival under nutrient and oxygen depleted conditions has been discussed. This is followed by a general discussion on gene expression and regulation in the microbe. The physiology of bacteria under stresses from the view of the transcriptional regulation of specific genes has also been discussed. The scope and objective of the present study in M. smegmatis covered in the thesis has been considered at the end. The next chapter discusses the characterization of msdps promoter in vivo with the help of reporter gene assay technologies. With the advent of promoterless E. coli-mycobacterium shuttle vectors, activity assays can be easily performed to characterize unknown upstream putative promoter sequences of genes. Both the 1 kb upstream as well as a 200bp upstream region of msdps gene has been characterized by. Primer extension analysis and subsequent site directed mutagenesis studies reveal +1 transcription start site and the promoter consensus sequence for the msdps gene respectively. Next chapter comprises of the method of constructing heterologous in vitro transcription machinery in mycobacteria. It is followed by characterization of transcription initiation at two dps promoters of M. smegmatis. A novel pull-down assay has been designed which enabled us to identify the sigma factors in the reconstituted RNA polymerases to be associated with the respective dps promoters and to compare the regulation of the two genes at transcription level. Further characterization through single round in vitro transcription at mycobacterial promoters has been attempted. The following two chapters provide some newer insights into the structure-function relationship of the first Dps molecule, MsDps (MsDps1) with respect to its DNA binding activity. The DNA binding activity is associated with the higher oligomeric form only. With the help of time resolved anisotropy and Förster Resonance Energy Transfer (FRET) experiments, we have monitored the nature of Dps dodecamer-DNA complex and mapped the distance between the N and C169 position in the absence and the presence of DNA. A new computational programme, Maximum Entropy Method (MEM) has been applied successfully to analyze data obtained from phase-modulation (Phi-M) lifetime experiments in order to get distribution of lifetime. In the last chapter a new method is adopted to predict amino acids important for stabilizing the interface in a trimeric structure. Subsequently, single and double amino acid mutants of the native MsDps protein has been constructed through site directed mutagenesis and are scored for the ability of the mutants to oligomerize under conditions similar to that of the native protein. This helped us to propose a hypothetical model of the overall mechanism of the protein oligomerization process in solution.
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A functional genomics approach to map transcriptional and post-transcriptional gene regulatory networksBhinge, Akshay Anant 15 October 2009 (has links)
It has been suggested that organismal complexity correlates with the complexity
of gene regulation. Transcriptional control of gene expression is mediated by binding of
regulatory proteins to cis-acting sequences on the genome. Hence, it is crucial to identify
the chromosomal targets of transcription factors (TFs) to delineate transcriptional
regulatory networks underlying gene expression programs. The development of ChIP-chip
technology has enabled high throughput mapping of TF binding sites across the
genome. However, there are many limitations to the technology including the availability
of whole genome arrays for complex organisms such human or mouse. To circumvent
these limitations, we developed the Sequence Tag Analysis of Genomic Enrichment
(STAGE) methodology that is based on extracting short DNA sequences or “tags” from
ChIP-enriched DNA. With improvements in sequencing technologies, we applied the
recently developed ChIP-Seq technique i.e. ChIP followed by ultra high throughput
sequencing, to identify binding sites for the TF E2F4 across the human genome. We identified previously uncharacterized E2F4 binding sites in intergenic regions and found
that several microRNAs are potential E2F4 targets.
Binding of TFs to their respective chromosomal targets requires access of the TF
to its regulatory element, which is strongly influenced by nucleosomal remodeling. In
order to understand nucleosome remodeling in response to transcriptional perturbation,
we used ultra high throughput sequencing to map nucleosome positions in yeast that were
subjected to heat shock or were grown normally. We generated nucleosome remodeling
profiles across yeast promoters and found that specific remodeling patterns correlate with
specific TFs active during the transcriptional reprogramming.
Another important aspect of gene regulation operates at the post-transcriptional
level. MicroRNAs (miRNAs) are ~22 nucleotide non-coding RNAs that suppress
translation or mark mRNAs for degradation. MiRNAs regulate TFs and in turn can be
regulated by TFs. We characterized a TF-miRNA network involving the oncofactor Myc
and the miRNA miR-22 that suppresses the interferon pathway as primary fibroblasts
enter a stage of rapid proliferation. We found that miR-22 suppresses the interferon
pathway by inhibiting nuclear translocation of the TF NF-kappaB. Our results show how
the oncogenic TF Myc cross-talks with other TF regulatory pathways via a miRNA intermediary. / text
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