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Chromatin-mediated regulation of the extracellular microenvironment in brain development and evolutionCubillos, Paula 06 March 2025 (has links)
Die Entwicklung komplexer Verhaltensweisen, welche das Entstehen von Kultur, Wissenschaft, Kunst und Literatur ermöglichten, wurden mit der Expansion des Neokortex im Laufe der menschlichen Evolution in Verbindung gebracht. Unterschiede in der Neuronenanzahl von Säugetieren sowie die einzigartige Intelligenz des Menschen wurden mit dieser Expansion assoziiert. Es gibt Hinweise darauf, dass Variationen in der Neuronenanzahl von Säugetieren durch Unterschiede in der proliferativen Kapazität von neuralen Vorläuferzellen (engl. NPCs) widergespiegelt werden. Daher kann die Untersuchung der Mechanismen, welche die Proliferation von NPCs fördern, dazu beitragen, die Expansion des menschlichen Neokortex besser zu verstehen. Differenzen in der proliferativen Kapazität von NPCs zwischen den Säugetierarten wurden mit dem Auftreten genetischer Neuheiten während der Evolution in Verbindung gebracht. Mehrere spezifisch menschliche Gene wurden als treibende Kräfte der Expansion des menschlichen Neokortex identifiziert. Dennoch wurde die Mehrheit der Veränderungen im menschlichen Genom in nicht-kodierenden Bereichen beschrieben, was darauf hindeutet, dass Veränderungen der Genregulations-Sequenzen sowie deren Auswirkung auf das Genexpressions-Muster an der Expansion des menschlichen Neokortex beteiligt sein könnten. Der transkriptomische Vergleich zwischen sich entwickelnden Gehirnen von Maus, einer Spezies mit kleinem Gehirn, und Mensch hat die Bedeutung der extrazellulären Nische während der Gehirnentwicklung hervorgehoben. Dennoch wurden Studien der Genregulations-Mechanismen von differentiell exprimierten Genen (engl. DEG), die mit der extrazellulären Nische in Verbindung gebracht werden, noch nicht beschrieben. In dieser Arbeit wurde nach DEG zwischen sich entwickelndem Neokortex von Maus und Mensch gesucht und drei Kandidatengene, namentlich Acan, Lamc2 und Ereg, auf Grund ihrer Assoziation mit der extrazellulären Nische, ausgewählt. Die Gene Acan und Lamc2 kodieren für zwei Proteine der extrazellulären Matrix (engl. ECM), AGGRECAN und die Gamma-2- Untereinheit von LAMININ-5, während Ereg für den Wachstumsfaktor EPIREGULIN kodiert. Für alle drei Gene ist die Expression während der menschlichen, aber nicht der murinen Gehirnentwicklung vorhanden. In dieser Studie wurden alle drei Proteine, AGGRECAN, LAMININ-5 und EPIREGULIN, untersucht, um ihre Rolle bei der Proliferation von NPCs zu bestimmen. Allerdings zeigte nur EPIREGULIN, dass es die Proliferation von NPCs durch den epidermalen Wachstumsfaktorrezeptor (engl. EGFR) im sich entwickelnden Neokortex der Maus förderte. Zusätzlich zeigte die Ablation von EREG in menschlichen kortikalen Organoiden eine Verringerung der NPC-Proliferation und der Neuronenbildung. Interessanterweise förderte EPIREGULIN die Proliferation von NPCs in Gorilla-, nicht aber in menschlichen kortikalen Organoiden. Schließlich identifizierten wir potenzielle Enhancer-Regionen in der Nähe des menschlichen EREG-Gens, die an der Regulation der EREG-Expression während der Neokortexentwicklung beteiligt sein könnten. Zusammenfassend legt diese Studie nahe, dass inter-spezies Unterschiede in der EREG-Expression, vermittelt durch Chromatin-Regulation, die Proliferation von NPCs während der Gehirnentwicklung fördern könnte, was zu einer erhöhten Neuronenbildung und zur Expansion des Neokortex beitragen haben könnte.
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Differentielle Genexpressionsanalyse aktivierter Endothelzellen / Differential genexpression-analysis of activated endothelial cellsSchmidt, Tobias 30 April 2001 (has links)
No description available.
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L’immunoprotéasome : régulateur de transcription et promoteur de survie cellulaireRouette, Alexandre 04 1900 (has links)
Le protéasome (CP) contrôle la majorité des fonctions cellulaires par la dégradation des protéines intracellulaires. En plus d’exprimer le CP, les vertébrés expriment également l’immunoprotéasome (IP), caractérisé par des préférences de dégradation distinctes. Le rôle le mieux caractérisé pour l’IP est la génération d’antigènes adaptés pour la liaison au complexe majeur d’histocomptabilité de classe I (CMH-I). Cependant, les nombreux phénotypes observés au niveau de cellules déficientes en IP ou avec une mutation révèlent que l’IP influence des fonctions immunitaires indépendamment de la génération d’antigènes et peut atténuer le stress présent au niveau de cellules non-immunitaires. L’objectif de cette thèse était de caractériser les rôles de l’IP qui ne sont pas reliés à la génération d’antigènes associés au CMH-I. L’analyse du transcriptome de cellules dendritiques IP-déficientes en cours de maturation révèle que l’IP affecte l’expression de plus de 8 000 transcrits. L’IP affecte l’expression génique principalement au niveau transcriptionnel en contrôlant l’abondance de régulateurs de transcriptions tels que NF-κB et les membres des familles IRF et STAT. Les cellules dendritiques IP-déficientes sont également moins efficaces pour activer des lymphocytes T CD8+, même chargées artificiellement avec des quantités optimales d’antigènes associés au CMH-I. En outre, nos études montrent que l’IP est fortement exprimé au niveau de cellules de patients atteints de leucémie myéloïde aigue. L’expression de l’IP est intrinsèque aux leucémies, puisque qu’elle n’est pas corrélée à la présence de lymphocytes sécréteurs d’IFN-γ. De plus, l’expression d’IP est particulièrement élevée au niveau de leucémies monocytaires et/ou possédant un réarrangement MLL. Notamment, des analyses de corrélation montrent que l’IP est connecté à des gènes impliqués dans le métabolisme, l’activité mitochondriale et la réponse au stress. En effet l’inhibition de la sous-unité PSMB8 de l’IP mène à l’accumulation de protéines ubiquitinées et la mort de cellules leucémiques monocytaires. Globalement, nos travaux montrent que le rôle de l’IP n’est pas limité à la génération d’antigènes, mais qu’il peut contrôler l’expression génique et la survie des leucémies. / By regulating protein degradation, constitutive proteasomes (CP) control practically all cellular functions. In addition to CP, vertebrates express immunoproteasomes (IP), which display distinct substrate preferences. The first non-redundant role ascribed to IP is its enhanced ability to generate MHC I-associated antigens. However, deletion or inhibition of IP subunits can affect several immune cell functions independently of MHC-I antigen generation. Moreover, recent work has shown that IP can be expressed in non-immune cells to deal with cell stress. Thus, we wished to investigate the roles of IP that are not related to antigen generation and that are not redundant with the CP. Based on profiling of WT and IP-deficient maturing mouse dendritic cells (DCs), we report that IP regulate the expression of more than 8,000 transcripts. The broad impact of IP on gene expression is cell-autonomous, mediated mainly at the transcriptional level, and involves major signaling pathways including IRFs, NF-kB and STATs. Moreover, even when engineered to present optimal amounts of antigenic peptides, IP-deficient DCs are inefficient for in vivo T-cell priming. In addition, consistent with the fact that cancer cells endure proteotoxic stress, we report that acute myeloid leukemia (AML) cells from patients express high levels of IP genes. Expression of IP genes in AML is a cell-autonomous and IFN-independent feature that correlates with the methylation status of IP genes, and is particularly high in AML with a monocytic phenotype and/or MLL rearrangement. Notably, IP inhibition leads to accumulation of polyubiquitinated proteins and cell death in IPhigh but not IPlow AML cells. Co-clustering analysis reveals that genes correlated with IP subunits in monocytic AMLs are primarily implicated in cell metabolism and proliferation, mitochondrial activity and stress responses. Overall, our studies show that the role of IP is not limited to antigen processing and reveals major non-redundant roles for IP in transcription regulation and resistance to cell stress in AML.
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Modulation de l’expression du gène CFTR par le produit du gène FIC1 responsable de la cholestase familiale intra-hépatique progressive de type 1 : Identification des mécanismes moléculaires impliquésSergent, Jacques-Aurélien 05 1900 (has links)
Réalisé en cotutelle avec l'Université de Cergy-Pontoise / La cholestase intra-hépatique familiale progressive de type 1 (PFIC1) humaine est une
maladie génétique rare, provoquée par des mutations du gène ATP8B1, due à un défaut de
sécrétion des acides biliaires. Un syndrome moins sévère, et épisodique, appelé Cholestase
Intra-hépatique Récurrente Bénigne (BRIC) a pu être associé à des mutations au sein du
même gène. Les patients PFIC1 souffrent de nombreuses manifestations extra-hépatiques.
Certaines de ces manifestations sont communes aux patients mucoviscidosiques. Le niveau
d’expression de CFTR, gène responsable de la mucoviscidose, est diminué chez les patients
PFIC1.
Cette étude a porté sur l’analyse des interactions/régulations entre CFTR et ATP8B1.
Une première approche a été de montrer l’expression de ces gènes dans différentes lignées
cellulaires puis d’identifier la présence de leurs protéines par western blot et
immunofluorescence. Une seconde approche a été d’effectuer une analyse in silico de la
structure d’ATP8B1 par rapport à sa fonction. Nous avons aussi localisés les modifications
connues sur un modèle 2D. Cette analyse a permis de mettre en évidence en plus des sites
connus (ATPase et domaines transmembranaires), deux sites de maturations par clivage
ainsi qu’un domaine riche en phosphorylation, des domaines PDZ et un domaine
d’interaction avec des récepteurs nucléaires et des facteurs de transcription. A partir d’un
polypeptide de 180 kDa, le clivage au niveau des sites identifiés produit un peptide de 145
kDa puis un de 90 kDa, révélés par western blot avec un anticorps dirigé contre la partie CTerminale
de la protéine. Ce peptide de 90 kDa, après myristoylation, pourrait interagir avec
des récepteurs nucléaires et des facteurs de transcriptions. Ces interactions nous ont permis
de monter un modèle qui pourrait expliquer la diminution d’expression génique de
différents gènes observés chez les malades PFIC1. Cette analyse a été poursuivie par une
étude de l’interactome d’ATP8B1 qui a montré une interaction possible avec CFTR
directement ou par l’intermédiaire d’une protéine de liaison, PDZK1. Une dernière étude a
porté sur la fonctionnalité de CFTR dans deux lignées portant des mutations différentes
d’ATP8B1. L’ensemble des résultats montre qu’ATP8B1 participerait à la régulation de
l’expression du gène CFTR mais aussi à sa maturation fonctionnelle. / Human Progressive Familial Intrahepatic Cholestasis type 1 (PFIC1) is a rare genetic
disease provoked by mutations inside the ATP8B1 gene resulting in a general loss of bile
acids secretion. An episodic and less severe syndrome called Benign Recurrent Intrahepatic
Cholestasis (BRIC) have also been associated with mutations in this gene. PFIC1 patients are
suffering from many extra-hepatic manifestations. Some of these manifestations are
common to Cystic Fibrosis (CF) patients, carrying mutations in CFTR gene. Moreover,
expression of CFTR is decreased for some PFIC1 patients.
This study was carried out to define the role of ATP8B1 in the modulation of CFTR
gene expression and protein function. A first approach was to identify both gene expression
and protein synthesis among various cell lines. Then, we developed a second approach
based on in silico analysis of structure and function of ATP8B1 to construct a 2D model of the
protein. This approach was correlated with the localization of known mutations of ATP8B1.
This analysis showed two possible protein maturation sites, a rich phosphorylation domain
and a nuclear receptor interacting domain. The cleavage of the 180 kDa peptide generates a
145kDa (ATPase) and a second cleavage produces a 90 kDa, all identified with a specific
antibody directed toward the C-Terminal region of the protein. The 90 kDa peptide should
be readdressed to the nucleus after myristoylation to interact with nuclear receptors and
transcription factors. This analysis was completed by an interactomic approach which has
shown a possible interaction between CFTR and ATP8B1 proteins either directly or mediated
by a linker, PDZK1. The last part of this work was dedicated to assess the role of ATP8B1 on
the activity of CFTR using two cell lines expressing two different mutated ATP8B1 genes.
From all these results, we concluded that ATP8B1 is probably involved in the regulation of
CFTR gene expression and CFTR maturation and function. We therefore propose a schematic
representation of ATP8B1 synthesis and maturation associated with its putative biological
functions in the cell.
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Les récepteurs intracellulaires de l'angiotensine II : nouvelles cibles thérapeutiques pour le remodelage cardiaqueTadevosyan, Artavazd 04 1900 (has links)
L'angiotensine-II (Ang-II), synthétisée à partir de sources extracardiaques et intracardiaques, régule l'homéostasie cardiaque en favorisant des effets mitogéniques et en promouvant la croissance cellulaire résultant d’une altération de l'expression génique. Dans cette étude, nous avons évalué la possibilité que les récepteurs de l'angiotensine-1 (AT1) ou les récepteurs de l'angiotensine-2 (AT2) situés sur l'enveloppe nucléaire régulent l’expression génique des cardiomyocytes. En analysant les noyaux cellulaires retenus des fractions de cœur de rat par immunobuvardage Western, nous avons détecté une co-purification préférentielle des protéines AT1 et AT2 avec un marqueur de la membrane nucléaire (Nup 62), par rapport aux marqueurs de la membrane plasmique (Calpactin I), de l’appareil de Golgi (GRP 78) ou du réticulum endoplasmique (GM130). La microscopie confocale a permis de démontrer la présence des AT1 et AT2 dans les membranes nucléaires. La microinjection de l’Ang-II-FITC sur des cardiomyocytes a provoqué une liaison de préférence aux sites nucléaires. Les enregistrements de transients calciques ont illustré que les AT1 nucléaires régulent le relâchement du Ca2+. L’incubation des ligands spécifiques d’AT1 et d’AT2 avec l’UTP [α32P] a résulté en une synthèse de novo d’ARN (par exemple, 16,9 ± 0,5 cpm/ng ADN contrôle vs 162,4 ± 29,7 cpm/ng ADN-Ang II, 219,4 ± 8,2 cpm/ng ADN L -162313 (AT1) et 126,5 ± 8,7 cpm/ng ADN CGP42112A (AT2), P <0,001). L’incubation des noyaux avec Ang-II augmente de façon significative l’expression de NFκB, une réponse qui est réprimée partiellement par la co-administration de valsartan ou de PD123177. Les expériences dose-réponse avec Ang-II administrée à l'ensemble des noyaux purifiés vs. aux cardiomyocytes seuls a montré une augmentation plus importante dans les niveaux d'ARNm de NFκB avec une affinité de ~ 3 fois plus grande (valeurs d’EC50 = 9 contre 28 pmol/L, respectivement), suggérant un rôle préférentiel nucléaire dans la signalisation. Par conséquent, nous avons conclu que les membranes cardiaques nucléaires possèdent des récepteurs d’Ang-II couplés à des voies de signalisation et à la transcription génique. La signalisation nucléaire pourrait jouer un rôle clé dans les changements de l'expression de gènes cardiaques, entraînant ainsi des implications mécanistiques et thérapeutiques diverses. / Angiotensin-II (Ang-II) from extracardiac sources and intracardiac synthesis regulates cardiac homeostasis, with mitogenic and growth-promoting effects largely due to altered gene-expression. In this study, the possibility that angiotensin-1 (AT1R) or angiotensin-2 (AT2R) receptors are located on the nuclear envelope and mediate effects on cardiomyocyte gene expression was assessed. Western blot tests of nucleus-enriched rat heart fractions indicated the presence of AT1R and AT2R proteins that preferentially copurified with a nuclear membrane marker (Nup 62) but not markers of plasma (Calpactin I), Golgi apparatus (GRP 78) or endoplasmic reticulum (GM130) membranes. Confocal microscopy revealed the existence of AT1R and AT2R proteins on nuclear membranes. Microinjected Ang-II preferentially bound to nuclear sites of isolated cardiomyocytes. Ca2+i-recordings on nuclear preparations demonstrated an AT1R-mediated Ca2+ release. AT1R and AT2R ligands enhanced de novo RNA synthesis in isolated cardiomyocyte nuclei incubated with [α32P]UTP (e.g. 16.9 ± 0.5 cpm/ng for DNA control vs. 162.4 ± 29.7 cpm/ng for DNA Ang-II, 219.4 ± 8.2 cpm/ng for DNA L-162313 (AT1) and 126.5 ± 8.7 cpm/ng for DNA CGP42112A (AT2), P<0.001). Ang-II application to isolated cardiomyocyte nuclei enhanced NFκB mRNA-expression, a response that was suppressed by co-administration of valsartan or PD123177. Dose-response experiments with Ang-II applied to purified nuclei vs. to whole cardiomyocytes showed a greater increase in NFκB mRNA levels at saturating concentrations with ~3 fold greater affinity (EC50 values 9 vs. 28 pmol/L, respectively), suggesting preferential nuclear signaling. These results lead us to conclude that cardiac nuclear membranes possess angiotensin receptors that couple to nuclear signaling pathways and regulate transcription. Signaling within the nuclear envelope (e.g. from intracellularly synthesized Ang-II) may play a role in Ang-II-mediated changes in cardiac gene-expression, with potentially important mechanistic and therapeutic implications.
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Étude de la fonction du promoteur foetal A[gamma] dans la régulation de la commutation de l'hémoglobine foetale à adulteBeauchemin, Hugues January 2008 (has links)
Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.
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Statistical analysis of networks and biophysical systems of complex architecture / L'analyse statistique des réseaux et des systèmes biophysiques de l'architecture complexeValba, Olga 15 October 2013 (has links)
De nombreux systèmes biologiques présentent une organisation complexe. Par exemple, les biopolymères peuvent posséder une structure très hiérarchisée responsable de leur fonction particulière. Comprendre la complexité de cette organisation permet de décrire des phénomènes biologiques et de prédire les fonctions des molécules. En outre, en supposant que la structure primaire du polymère est formée aléatoirement, nous pouvons essayer de caractériser ce phénomène par des grandeurs probabilistes (variances, moyennes, etc). Cette formulation est propre aux problèmes d'évolution.Les réseaux biologiques sont d'autres objets communs de la physique statistique possédant de riches propriétés fonctionnelles. Pour décrire un mécanisme biologique, on utilise différents types de réseaux biomoléculaires. Le développement de nouvelles approches peut nous aider à structurer, représenter et interpréter des données expérimentales, comprendre les processus cellulaires et prédire la fonction d'une molécule.L'objectif de cette thèse est de développer des méthodes pour l'étude d'objets statiques ou dynamiques, ayant une architecture complexe. Ici, nous nous intéressons à deux problèmes.La première partie est consacrée à l'analyse statistique des biopolymères aléatoires. Nous étudions une transition de phase présente dans les séquences aléatoires de l'ARN. On met alors en évidence deux modes : le régime où presque toutes les bases qui composent l'ARN sont couplées et la situation où une fraction finie de ces bases restent non complémentaires.La deuxième partie de cette thèse se concentre sur les propriétés statistiques des réseaux. Nous développons des méthodes pour l'identification d'amas de gènes co-expressifs sur les réseaux et la prédiction de gènes régulateurs novateurs. Pour cela, nous utilisons la fonction du plus court chemin et l'analyse du profil des motifs formés par ces amas. Ces méthodes ont pu prédire les facteurs de transcription impliqués dans le processus de longévité. Enfin, nous discutons de la formation de motifs stables sur les réseaux due à une évolution sélective. / Complex organization is found in many biological systems. For example, biopolymers could possess very hierarchic structure, which provides their functional peculiarity. Understating such, complex organization allows describing biological phenomena and predicting molecule functions. Besides, we can try to characterize the specific phenomenon by some probabilistic quantities (variances, means, etc), assuming the primary biopolymer structure to be randomly formed according to some statistical distribution. Such a formulation is oriented toward evolutionary problems.Artificially constructed biological network is another common object of statistical physics with rich functional properties. A behavior of cells is a consequence of complex interactions between its numerous components, such as DNA, RNA, proteins and small molecules. Cells use signaling pathways and regulatory mechanisms to coordinate multiple processes, allowing them to respond and to adapt to changing environment. Recent theoretical advances allow us to describe cellular network structure using graph concepts to reveal the principal organizational features shared with numerous non-biological networks.The aim of this thesis is to develop bunch of methods for studying statistical and dynamic objects of complex architecture and, in particular, scale-free structures, which have no characteristic spatial and/or time scale. For such systems, the use of standard mathematical methods, relying on the average behavior of the whole system, is often incorrect or useless, while a detailed many-body description is almost hopeless because of the combinatorial complexity of the problem. Here we focus on two problems.The first part addresses to statistical analysis of random biopolymers. Apart from the evolutionary context, our studies cover more general problems of planar topology appeared in description of various systems, ranging from gauge theory to biophysics. We investigate analytically and numerically a phase transition of a generic planar matching problem, from the regime, where almost all the vertices are paired, to the situation, where a finite fraction of them remains unmatched.The second part of this work focus on statistical properties of networks. We demonstrate the possibility to define co-expression gene clusters within a network context from their specific motif distribution signatures. We also show how a method based on the shortest path function (SPF) can be applied to gene interactions sub-networks of co-expression gene clusters, to efficiently predict novel regulatory transcription factors (TFs). The biological significance of this method by applying it on groups of genes with a shared regulatory locus, found by genetic genomics, is presented. Finally, we discuss formation of stable patters of motifs in networks under selective evolution in context of creation of islands of "superfamilies".
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Caracterização de novos genes humanos envolvidos no processo de regulação da expressão de genes homeóticos / Characterization of novel human genes involved in the regulation of expression of homeotic genesNunes, Diana Noronha 03 September 2004 (has links)
A identidade na segmentação do corpo de diversos organismos, durante o desenvolvimento, é devida, em grande parte, à ação das proteínas homeóticas. Em especial, dois grupos de proteínas, Trithorax (trxG) e Polycomb (PcG) têm um papel fundamental na manutenção, respectivamente, da ativação e da repressão da transcrição gênica, associando-se à cromatina. A importância das PcG nos estimulou a buscar a caracterização das proteínas humanas ortólogas ao \"Enhancer of Polycomb\" (Epc) de Drosophila, até então não descritas no genoma humano. Para tanto, buscamos: - obter a sequência completa e mapear o cDNA do novo gene humano homólogo ao \"Enhancer of Polycomb\" de Drosophila; - analisar sua expressão em tecidos fetais, adultos e tumorais e fazer estudos buscando sua caracterização funcional. Encontramos, mapeamos e obtivemos a seqüência completa de dois genes humanos, ortólogos de Epc1 (10p11-22) e de Epc2 (2q21-23) de camundongo, publicando estes dados em 2001 (Camargo et al., 2001). Ambos os genes são bastante conservados entre várias espécies, sendo que o cDNA de hEPC2 humano, por exemplo, é 94% idêntico ao Epc2 de camundongo e possui 96% de identidade ao nível de proteína, sugerindo que a função do gene deve ter sido mantida durante a evolução. No entanto, as seqüências protéicas de hEPC1 e hEPC2 humanos possuem apenas 68% de identidade entre si. Portanto, é provável que após a duplicação dos parálogos, estes tenham divergido funcionalmente. A expressão de ambos os genes foi avaliada utilizando \"dot-blots\" contendo 76 mRNAs de amostras de tecidos fetais, adultos e tumorais, mostrando-se fraca e ubíqua. Análises in silico sugeriram a existência de 4 isoformas de splicing para hEPC2, as quais foram validadas por RT-PCR ou \"Northern blots\". Uma das isoformas (de 2.7 Kpb) se mostrou mais abundante em todas as linhagens tumorais estudadas através de análises de \"Northern blot\", principalmente nas linhagens de linfoma de Burkitt\'s Raji e na linhagem de leucemia pró-mielocítica HL-60. Esta isoforma é gerada através de um sítio alternativo de poli-adenilação, que reduz sua porção 3\'UTR, retirando 4 dos 5 \"elementos ricos em adenilatos e uridilatos\" (AREs), envolvidos com a degradação de mRNAs lábeis que codificam proteínas regulatórias. Estes resultados se encontram em um manuscrito recentemente submetido à publicação (anexo à tese). Interação entre hEPC2 e SMADs e sua modulação por TGF-β. Durante a montagem da seqüência completa de hEPC2, verificamos que duas ESTs patenteadas mostravam alta identidade com o gene. Estas seqüências foram descritas como sendo parte de uma nova proteína de interação com as proteínas da família SMAD, envolvidas com transdução de sinais desencadeados por TGF-β. Esta citocina por sua vez, regula a proliferação, diferenciação e morte celular. Partimos para a avaliação da possível interação entre hEPC2 e as SMADs, em colaboração com o grupo do Dr. Aristidis Moustakas, do Ludwig Institute for Cancer Research de Uppsala, Suécia. Os resultados de co-imunoprecipitação sugeriram que as SMADs 2, 3, 4, 7 e 8 interagem com hEPC2, sendo que a interação entre as SMAD2, SMAD3, SMAD4 e hEPC2 nas células tratadas com TGF-β1, mostraram uma redução na co-imunoprecipitação. Este resultado sugere que TGF-β1 modula negativamente a interação entre essas proteínas. Da mesma maneira, foi observada uma redução na interação de hEPC2 com SMAD8 após o tratamento com BMP-7. Esse resultado é ainda mais destacado para as SMADs 2 e 3. Estes dados foram observados para ambas as construções de hEPC2, o que sugere fortemente a veracidade da interação entre estas proteínas. A localização celular de hEPC2, e também sua co-localização com SMAD2 foram investigadas através de imunofluorescência indireta e confirmaram a predição do programa PSORTII, de que hEPC2 se localiza no núcleo. No entanto, não foi possível observar a co-localização entre hEPC2 e SMAD2. É possível que hEPC2 não se ligue diretamente ao DNA, necessitando se associar como parceiro de um fator de transcrição. Esta foi uma das hipóteses para a atuação de hEPC2, como um co-fator que se associe com uma das SMADs e se ligue a um elemento específico de ligação a SMAD (SBE). Para investigar essa hipótese um ensaio de gene repórter foi feito utilizando uma construção de um repórter contendo 12 repetições da seqüência CAGA (seqüência específica de ligação das SMADs 2,3 e 4) fusionado com o gene da luciferase. No entanto, este ensaio não demonstrou que a transcrição de SMAD2 é dependente de hEPC2 e o experimento deverá ser repetido. Para confirmar a interação entre hEPC2 e as SMADs, será feito um experimento de \"pull-down\". Para tal o cDNA de hEPC2 foi clonado no vetor pET-32A de expressão indutível em bactérias. A proteína recombinante já foi produzida, tendo sido induzida e posteriormente purificada em condições desnaturantes. Apesar de dezenas de genes PcG terem sido caracterizados em Drosophila, poucos destes genes foram estudados em mamíferos. Portanto, a descrição do gene hEPC2 e seus transcritos alternativos, contribui para o conhecimento de PcG humanos, indicando a associação de maior expressão de uma de suas isoformas em linhagens celulares tumorais. Em relação à interação de hEPC2 com as SMADs, é interessante observar que nenhuma outra proteína foi descrita por possuir a particularidade de interagir com as SMADs de diferentes categorias. Talvez este seja um dado importante, que indique o papel singular de hEPC2 na sinalização de TGF-β1. / The identity of body segmentation in several organisms during development is, to a large extent, due to the action of the homeotic proteins. In particular, two groups of proteins, the Trithorax (trxG) and Polycomb (PcG), have a major role in maintenance of respectively, transcription activation and repression, when associated to the chromatin. The importance of PcGs has motivated us to pursue the isolation and characterization of two new human proteins that are orthologs of the \"Enhancer of Polycomb\" (Epc) of Drosophila. To achieve this goal we undertook the task of the cloning and mapping of complete cDNA sequence of the novel genes hEPC1 and hEPC2, analyzing its expression in fetal, adult and tumoral tissues and functionally characterizing the hEPC2 protein. In 2001, we published the mapping and cloning of the complete cDNA sequences of both genes, as being orthologs of the mouse Epc1 (10p11-22) and Epc2 (2q21-23), together with the strategy used to obtain the full-length cDNAs (Camargo et al., 2001). Both genes are shown to be highly conserved among several species. Thus, the human hEPC2 cDNA is 94% identical to the mouse Epc2 and displays 96% identity at the protein level, suggesting maintenance of its function during the evolution. However, the protein sequences of the human hEPC1 and hEPC2 display only 68% identity. Therefore, it is likely that they have undergone a functional divergence after their duplication. The expression of both genes was evaluated using \"dot-blots\" containing 76 mRNAs samples from fetal, adult and tumoral tissues and is shown to be weak and ubiquitous. \"In silico\" analysis suggested the existence of 4 hEPC2 splicing isoforms that were validated by RT-PCR and/or Northern-blots. One of the isoforms (of 2.7 Kbp) is shown to be more abundant in all of the tumoral cell lines evaluated using Northern-blot analysis, mainly in the Burkit\'s Raji lymphoma and in the promyelocytic leukemia HL-60. This isoform results from the use of an alternative polyadenylation site that reduces the 3\'UTR, abolishing 4 of 5 \"adenylates and urilates rich elements\" (AREs), involved in the degradation of labile mRNAs that codify to regulatory proteins. These results have been recently submitted to publication (manuscript attached to this thesis). Interaction between the hEPC2/SMADs and its modulation by TGF-β. During the assembly of the hEPC2 full-length cDNA sequence, we found two patented ESTs that tagged a portion of the gene. These sequences were described as partial sequences of a \"new SMAD interacting protein\", involved in signal transduction of TGF-β, a cytokine that regulates cell proliferation, differentiation and death. To evaluate this putative interaction between hEPC2 and the SMADs proteins, we begun a collaboration with the TGF-β signalling group of the Dr. Aristidis Moustakas, from the Uppsala Ludwig Institute for Cancer Research, Sweden. The results of co-imunoprecipitation assays suggested that SMADs 2, 3, 4, 7 e 8 interact with hEPC2. Moreover, the interaction among SMAD2, SMAD3, SMAD4 and hEPC2 in cells treated with TGF-β1 showed decreased co-imunoprecipitation. This result suggests that TGF-β1 negatively modulates the interaction of these proteins. Likewise, we observed a reduction in hEPC2 interaction with SMAD8 upon BMP-7 treatment. This effect was even more dramatic for SMADs 2 and 3. These data were observed for both hEPC2 plasmid constructs, which strongly suggest the veracity of these proteins interaction. The cell localization of the hEPC2 protein, as well as its co-localization with the SMAD2, were investigated through indirect immunofluorescence assay, confirming the predicted localization of hEPC2 in the cell nucleus using the PSORTII program. However, we were not able to confirm the co-localization of hEPC2 and SMAD2. It is possible that hEPC2 does not bind directly to the DNA, requiring an association with a partner such as a transcription factor. This raises the hypothesis of hEPC2 having a role as a co-factor associated to one of the SMADs and binding to a \"SMAD binding element\" (SBE). To investigate this hypothesis, gene reporter assays were undertaken using a reporter construct containing 12 CAGA sequence repetitions (specific binding sequence of the SMADs 2, 3 and 4) fused to the luciferase gene. However, this assay could not demonstrate that the transcription of the SMAD is dependent on hEPC2. This experiment must be repeated. To confirm the interaction of hEPC2 and SMADs, a pull-down assay will be performed. To this end, the coding region of hEPC2 was cloned into the pET-32A bacterial inducible expression vector. The recombinant protein was already produced, having been induced and purified under denaturing conditions. Despite the dozens of PcG genes that were described in Drosophila, only a few of these genes have been characterized in mammals. Therefore, the description of the hEPC2 and its alternative transcripts is a contribution to better knowledge of the human PcGs. Regarding the hEPC2 and SMADs interaction, it\'s it is noteworthy that this is the first protein described to interact with SMADs of distinct categories. This may be an important indication of a unique role for hEPC2 in the TGF-β1 signaling pathway.
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Estudo da RNA helicase DEAD-box codificada pelo gene CC0835 em Caulobacter crescentus. / Study of the DEAD-box RNA helicase encoded by the gene CC0835 in Caulobacter crescentus.Vicente, Alexandre Magno 17 February 2017 (has links)
Com a construção da linhagem RlhE fusionada a um epitopo, fomos capazes de investigar o acúmulo da proteína após choque frio, em fase estacionária, durante o ciclo celular de Caulobacter crescentus, sob diferentes condições de estresses e, finalmente, após a adição de cloranfenicol, para o estudo a estabilidade protéica. Foi observado um aumento no acúmulo da proteína após choque frio. Além disso, quando em diferentes condições de estresses, RlhE obteve uma leve indução na presença de NaCl e Sacarose; mas permanaceu constante em fase estacionária e durante o ciclo celular. Finalmente, vimos que a estabilidade de RlhE varia de acordo com a temperatura, tendo um aumento da estabilidade a 10°C. As análises de expressão do gene rhlE foram realizadas por ensaios de atividade de betagalactosidase. Demonstramos que a presença da região 5UTR é importante para a indução, e que rlhE possui, pelo menos em parte, uma regulação pós-transcricional. Uma análise transcriptômica global da linhagem selvagem e mutante para rhlE, após o choque frio, foi realizada por RNAseq, o qual nos auxiliou na identificação de genes envolvidos em diversos processos biológicos. Finalmente, a co-imunoprecipitação e identificação dos RNAs por sequenciamento em larga escala revelou que RlhE interage com 51 mRNAs. / Here, we constructed a strain in which the RhIE protein was fusioned to an epitope that allowed the investigation of the protein profile after cold-shock, at stationary phase, during cell cycle of Caulobacter crescentus, under different environmental stresses, and, finally, after chloramphenicol addition to study protein stability. The results showed an increase in protein concentration after cold shock stress. When exposed to different stresses RhlE was slightly induced in the presence of NaCl and Sucrose; but its abundance remained constant at stationary phase and during C. crescentus cell cycle. Lastly, the protein stability varies depending on temperature - increasing at low temperature. Gene expression analysis was performed using beta-galactosidase assays. We showed that the presence of the 5UTR is important for the induction of rhlE, and that RhlE is posttranscriptionally regulated. A global transcriptome analysis was performed using RNAseq after cold shock stress of the wild type strain and null mutant for rhlE, and several genes involved in a wide range of biological process were identified. Finally, High-throughput sequencing of RNA isolated by crosslinking immunoprecipitation revealed interactions of RhlE with 51 mRNAs.
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Regulação da adesão de Escherichia coli enteropatogênica (EPEC) por genes de resposta à limitação nutricional e estresse. / Regulation of enteropathogenic Escherichia coli (EPEC) adhesion by genes related to nutrional shortage and stress.Ferreira, Gerson Moura 24 August 2009 (has links)
Escherichia coli enteropatogênica (EPEC) é uma das principais causas de diarreia em crianças. Na carência de fosfato (Pi), um conjunto de genes conhecido como regulon PHO é induzido. Esse regulon é controlado pelo sistema Pst, que além de ser um transportador de Pi, reprime a expressão de PHO quando Pi é abundante, e pelo sistema de dois componentes PhoB/PhoR. A deleção de pst reduziu a adesão de EPEC à células epiteliais in vitro, pois diminuiu da expressão dos reguladores PerA/PerC, que por sua vez controlam a expressão de genes envolvidos na adesão. Este efeito foi exclusivo de pst e não devido a expressão constitutiva dos genes de PHO causada pela deleção de pst. A expressão da fímbria BFP, PerA e PerC também dependem da síntese de ppGpp, uma molécula de alarme envolvida na regulação de genes relacionados à carência nutricional. ppGpp regula positivamente a expressão de PerA e PerC. Entretanto, RpoS, o fator relacionado à resposta ao estresse, afetou negativamente o nível de adesão de EPEC e a expressão de BFP. / Enteropathogenic E. coli (EPEC) is one of the causes of diarrhea in children. Phosphate (Pi) shortage induces transcription of the genes known as the PHO regulon. These genes are controlled by the Pst system, that is also a high-affinity Pi transporter, and represses PHO expression under Pi-replete conditions. PHO is also controlled by the two-component system PhoB/PhoR. Deletion of the pst operon reduced the adhesion of EPEC to epithelial cells in vitro due to a decrease in the expression of the regulators PerA and PerC that in turn control the expression of genes related to adhesion. The constitutive expression of the PHO genes in the pst mutant was not the cause of adhesion inhibition. Expression of bfp and the regulators PerA and PerC was also dependent on ppGpp, an alarmone involved in the regulation of genes related to nutrient limitation. On the other hand, RpoS, the factor that controls the general stress response, negatively affected EPEC adhesion and bfpA expression.
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