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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Identification and functional characterisation of a PREP1-PBX protein complex

Berthelsen, Jens January 2000 (has links)
No description available.
2

Functional and structural properties of eukaryotic DNA polymerase epsilon

Chilkova, Olga January 2006 (has links)
In eukaryotes there are three DNA polymerases which are essential for the replication of chromosomal DNA: DNA polymerase alpha (Pol alpha), DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon). In vitro studies of viral DNA replication showed that Pol alpha and Pol delta are sufficient for DNA replication on both leading and lagging DNA strands, thus leaving the function of Pol epsilon unknown. The low abundance and the reported protease sensitivity of Pol epsilon were holding back biochemical studies of the enzyme. The aim of this study was to characterize the structural and functional properties of eukaryotic Pol epsilon. We first developed a protocol for over-expression and purification of Pol epsilon from the yeast Saccharomyces cerevisiae. Pol epsilon consists of four subunits: Pol2 (catalytic subunit), Dpb2, Dpb3 and Dpb4. This four-subunit complex was purified to homogeneity by conventional chromatography and the subunit stoichiometry of purified Pol epsilon was estimated from colloidal coomassie-stained gels to be 1:1:1:1. The quaternary structure was determined by sedimentation velocity and gel filtration experiments. Molecular mass (371 kDa) was calculated from the experimentally determined Stokes radius (74.5 Å) and sedimentation coefficient (11.9 S) and was in good agreement with a theoretical molecular mass calculated for a heterotetramer (379 kDa). Analytical sedimentation equilibrium ultracentrifugation experiments supported the proposed heterotetrameric structure of Pol epsilon. By cryo-electron microscopy and single-particle image analysis we determined the structure of Saccharomyces cerevisiae Pol epsilon to 20-Å resolution. The four-subunit complex was found to consist of a globular domain, comprising the Pol2 subunit, flexibly connected to an elongated domain, including Dpb2, Dpb3 and Dpb4 subunits. We found that Pol epsilon requires a minimal length of 40 base pairs of primer-template duplex to be processive. This length corresponds to the dimensions of the elongated domain. To characterize the fidelity by which Pol epsilon synthesizes DNA, we purified wild type and exonuclease-deficient Pol epsilon. Wild type Pol epsilon synthesizes DNA with a very high accuracy. Analysis of the exonuclease-deficient Pol epsilon showed that Pol epsilon proofreads more than 90% of the errors made by its polymerase activity. Exonuclease-deficient Pol epsilon was shown to have a specific spectrum of errors not seen in other DNA polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP and C-dTTP mispairs. This unique error specificity and amino acid sequence alignment suggest that the structure of the polymerase active site of Pol epsilon differs from those of other members of B family DNA polymerases. With recombinant proteins and circular single-stranded DNA templates, we partially reconstituted DNA replication in vitro, in which we challenged Pol epsilon and Pol delta in side-by-side comparisons regarding functional assays for polymerase activity and processivity, as well as physical interactions with nucleic acids and PCNA. We found that Pol epsilon activity and “on-DNA” PCNA interactions are dependent on RPA-coated template DNA. By the surface plasmon resonance technique, we showed that Pol epsilon has a high affinity for DNA and low affinity for immobilized PCNA. By contrast, Pol delta was found to have low affinity for DNA and high affinity for PCNA. We suggest that a possible function of RPA is to regulate down the DNA synthesis through Pol epsilon, and that the mechanism by which Pol epsilon and Pol delta load onto the template is different due to different properties of the interaction with DNA and PCNA.
3

Structural and Functional Evolution of Human Heat Shock Transcription Factors

Jaeger, Alex M. January 2015 (has links)
<p>Proteotoxic stress is implicated in numerous human diseases including neurodegeneration, cancer, and diabetes. Unfortunately, our mechanistic understanding of the cellular response to proteotoxic stress is limited. A critical feature of the cellular stress response is the activation of Heat Shock Transcription Factors (HSFs) that regulate the expression of numerous genes involved in protein folding, protein degradation, and cellular survival. The studies presented here utilize a diverse array of techniques including yeast genetics, recombinant protein expression and purification, biochemical analysis of protein-DNA interactions, x-ray crystallography, in vitro post-translational modification, and mammalian cell culture to illuminate novel aspects of HSF biology. Critical findings include understanding key principles of HSF-DNA interactions, identification of a novel negative regulator of HSF activity, and identification of structural features of HSF paralogs that enable precise combinatorial regulation. These unique insights lay the foundation for a greater understanding of HSF in specific cellular contexts and disease states.</p> / Dissertation
4

Large scale simulations of genome organisation in living cells

Johnson, James January 2018 (has links)
Within every human cell, approximately two meters of DNA must be compacted into a nucleus with a diameter of around ten micrometers. Alongside this daunting storage problem, the 3D organisation of the genome also helps determine which genes are up- or down-regulated, which in turn effects the functionality of the cell itself. While the organisational structure of the genome can be revealed using experimental techniques such as chromosome conformation capture and its high-throughput variant Hi-C, the mechanisms driving this organisation are still unclear. The first two results chapters of this thesis use molecular dynamics simulations to investigate the effect of a potential organisational mechanisms for DNA known as the "bridging-induced attraction". This mechanism involves multivalent DNA-binding proteins bridging genomically distant regions of DNA, which in turn promotes further binding of proteins and compaction of the DNA. In chapter 2 (the first results chapter) we look at a model where proteins can bind non-specifically to DNA, leading to cluster formation for suitable protein-DNA interaction strengths. We also show the effects of protein concentration on the DNA, with a collapse from a swollen to a globular phase observed for suitably high protein concentrations. Chapter 3 develops this model further, using genomic data from the ENCODE project to simulate the "specific binding" of proteins to either active (euchromatin) or inactive (heterochromatin) regions. We were then able to compare contact maps for specific simulated chromosomes with the experimental Hi-C data, with our model reproducing well the topologically associated domains (TADs) seen in Hi-C contact maps. In chapter 4 of the thesis we use numerical methods to study a model for the coupling between DNA topology (in particular, supercoiling in DNA and chromatin) and transcription in a genome. We present details of this model, where supercoiling flux is induced by gene transcription, and can diffuse along the DNA. The probability of transcription is also related to supercoiling, as regions of DNA which are negatively supercoiled have a greater likelihood of being transcribed. By changing the magnitude of supercoiling flux, we see a transition between a regime where transcription is random and a regime where transcription is highly correlated. We also find that divergent gene pairs show increased transcriptional activity, along with transcriptional waves and bursts in the highly correlated regime { all these features are associated with genomes of living organisms.
5

From network to pathway: integrative network analysis of genomic data

Wang, Chen 25 August 2011 (has links)
The advent of various types of high-throughput genomic data has enabled researchers to investigate complex biological systems in a systemic way and started to shed light on the underlying molecular mechanisms in cancers. To analyze huge amounts of genomic data, effective statistical and machine learning tools are clearly needed; more importantly, integrative approaches are especially needed to combine different types of genomic data for a network or pathway view of biological systems. Motivated by such needs, we make efforts in this dissertation to develop integrative framework for pathway analysis. Specifically, we dissect the molecular pathway into two parts: protein-DNA interaction network and protein-protein interaction network. Several novel approaches are proposed to integrate gene expression data with various forms of biological knowledge, such as protein-DNA interaction and protein-protein interaction for reliable molecular network identification. The first part of this dissertation seeks to infer condition-specific transcriptional regulatory network by integrating gene expression data and protein-DNA binding information. Protein-DNA binding information provides initial relationships between transcription factors (TFs) and their target genes, and this information is essential to derive biologically meaningful integrative algorithms. Based on the availability of this information, we discuss the inference task based on two different situations: (a) if protein-DNA binding information of multiple TFs is available: based on the protein-DNA data of multiple TFs, which are derived from sequence analysis between DNA motifs and gene promoter regions, we can construct initial connection matrix and solve the network inference using a constraint least-squares approach named motif-guided network component analysis (mNCA). However, connection matrix usually contains a considerable amount of false positives and false negatives that make inference results questionable. To circumvent this problem, we propose a knowledge based stability analysis (kSA) approach to test the conditional relevance of individual TFs, by checking the discrepancy of multiple estimations of transcription factor activity with respect to different perturbations on the connections. The rationale behind stability analysis is that the consistency of observed gene expression and true network connection shall remain stable after small perturbations are applied to initial connection matrix. With condition-specific TFs prioritized by kSA, we further propose to use multivariate regression to highlight condition-specific target genes. Through simulation studies comparing with several competing methods, we show that the proposed schemes are more sensitive to detect relevant TFs and target genes for network inference purpose. Experimentally, we have applied stability analysis to yeast cell cycle experiment and further to a series of anti-estrogen breast cancer studies. In both experiments not only biologically relevant regulators are highlighted, the condition-specific transcriptional regulatory networks are also constructed, which could provide further insights into the corresponding cellular mechanisms. (b) if only single TF's protein-DNA information is available: this happens when protein-DNA binding relationship of individual TF is measured through experiments. Since original mNCA requires a complete connection matrix to perform estimation, an incomplete knowledge of single TF is not applicable for such approach. Moreover, binding information derived from experiments could still be inconsistent with gene expression levels. To overcome these limitations, we propose a linear extraction scheme called regulatory component analysis (RCA), which can infer underlying regulation relationships, even with partial biological knowledge. Numerical simulations show significant improvement of RCA over other traditional methods to identify target genes, not only in low signal-to-noise-ratio situations and but also when the given biological knowledge is incomplete and inconsistent to data. Furthermore, biological experiments on Escherichia coli regulatory network inferences are performed to fairly compare traditional methods, where the effectiveness and superior performance of RCA are confirmed. The second part of the dissertation moves from protein-DNA interaction network up to protein-protein interaction network, to identify dys-regulated protein sub-networks by integrating gene expression data and protein-protein interaction information. Specifically, we propose a statistically principled method, namely Metropolis random walk on graph (MRWOG), to highlight condition-specific PPI sub-networks in a probabilistic way. The method is based on the Markov chain Monte Carlo (MCMC) theory to generate a series of samples that will eventually converge to some desired equilibrium distribution, and each sample indicates the selection of one particular sub-network during the process of Metropolis random walk. The central idea of MRWOG is built upon that the essentiality of one gene to be included in a sub-network depends on not only its expression but also its topological importance. Contrasted to most existing methods constructing sub-networks in a deterministic way and therefore lacking relevance score for each protein, MRWOG is capable of assessing the importance of each individual protein node in a global way, not only reflecting its individual association with clinical outcome but also indicating its topological role (hub, bridge) to connect other important proteins. Moreover, each protein node is associated with a sampling frequency score, which enables the statistical justification of each individual node and flexible scaling of sub-network results. Based on MRWOG approach, we further propose two strategies: one is bootstrapping used for assessing statistical confidence of detected sub-networks; the other is graphic division to separate a large sub-network to several smaller sub-networks for facilitating interpretations. MRWOG is easy to use with only two parameters need to be adjusted, one is beta value for performing random walk and another is Quantile level for calculating truncated posteriori mean. Through extensive simulations, we show that the proposed scheme is not sensitive to these two parameters in a relatively wide range. We also compare MRWOG with deterministic approaches for identifying sub-network and prioritizing topologically important proteins, in both cases MRWG outperforms existing methods in terms of both precision and recall. By utilizing MRWOG generated node/edge sampling frequency, which is actually posteriori mean of corresponding protein node/interaction edge, we illustrate that condition-specific nodes/interactions can be better prioritized than the schemes based on scores of individual node/interaction. Experimentally, we have applied MRWOG to study yeast knockout experiment for galactose utilization pathways to reveal important components of corresponding biological functions; we also applied MRWSOG to study breast cancer patient prognostics problems, where the sub-network analysis could lead to an understanding of the molecular mechanisms of antiestrogen resistance in breast cancer. Finally, we conclude this dissertation with a summary of the original contributions, and the future work for deepening the theoretical justification of the proposed methods and broadening their potential biological applications such as cancer studies. / Ph. D.
6

Discovery of New Protein-DNA and Protein-Protein Interactions Associated With Wood Development in Populus trichocarpa

Petzold, Herman E. III 09 November 2017 (has links)
The negative effects from rising carbon levels have created the need to find alternative energy sources that are more carbon neutral. One such alternative energy source is to use the biomass derived from forest trees to fulfill the need for a renewable alternative fuel. Through increased understanding and optimization of regulatory mechanisms that control wood development the potential exists to increase biomass yield. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA, normally located in the 5-prime upstream promoter region of the gene. In the first section of this work, six DNA promoters from wood formation-related genes were screened by the Yeast One-Hybrid (Y1H) assay in efforts to identify novel interacting TFs involved in wood formation. The promoters tested belong to genes involved in lignin biosynthesis, programmed cell death, and cambial zone associated TFs. The promoters were screened against a mini-library composed of TFs expressed 4-fold or higher in differentiating xylem vs phloem-cambium. The Y1H results identified PtrRAD1 with interactions involving several of the promoters screened. Further testing of PtrRAD1 by Yeast Two-Hybrid (Y2H) assay identified a protein-protein interaction (PPI) with poplar DIVARACATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay and formed PPIs with MYB/SANT domain proteins, homeodomain family (HD) TFs, and cytoskeletal-related proteins. In the second section of this work, PPIs involving PtrDRIF1s' interaction partners were further characterized. PtrDRIF1 is composed of two separate domains, an N-terminal MYB/SANT domain that interacted with the MYB/SANT domain containing PtrRAD1 and PtrDIVARICATA-like proteins, and a C-terminal region containing a Domain of Unknown Function 3755 (DUF3755). The DUF3755 domain interacted with HD family members belonging to the ancient WOX clade and Class II KNOX domain TFs. In addition, PtrDRIF1 was able to form a complex between PtrRAD1 and PtrWOX13c in a Y2H bridge assay. PtrDRIF1 may function as a regulatory module linking cambial cell proliferation, lignification, and cell expansion during growth. Combined, these findings support a role for PtrDRIF1 in regulating aspects of wood formation that may contribute to altering biomass yield. / Ph. D. / Trees are unique among plants since they have extremely long life spans and the ability to generate large quantities of woody biomass. The woody biomass derived from forest trees can function to provide renewable energy in the form of biofuels. The process of wood formation is complex and requires coordinated activation of genes involved in multiple metabolic pathways. Transcription factors (TFs) are DNA-binding regulatory proteins capable of either activation or repression by binding to a specific region of DNA. These protein-DNA interactions regulate gene expression during plant growth and development. In this study, new regulators of genes known to be involved in wood formation were identified using the Yeast One-Hybrid (Y1H) assay. One of the proteins identified, PtrRAD1 had not been previously linked to wood formation and was a candidate for further characterization. Further testing of PtrRAD1 by the Yeast Two-Hybrid (Y2H) assay resulted in identification of a protein-protein interaction with Populus trichocarpa DIVARICATA RADIALIS INTERACTING FACTOR (DRIF1). PtrDRIF1 was then used in the Y2H assay to identify numerous interacting proteins, in addition to those reported previously in other species. Further characterization of PtrDRIF1, identified an N-terminal region capable of forming interactions with MYB/SANT domain proteins, and C-terminal region that interacted with homeodomain proteins. PtrRAD1, PtrDRIF1, and the homeodomain containing PtrWOX13c were able to form a complex in an Y2H-bridge assay. Combined, these findings support a potential role for PtrDRIF1 in regulating wood polarity, wood formation, and stem cell proliferation.
7

Interação entre o fator de transcrição CG9571 e módulos reguladores do gene pair-rule even-skipped da cascata de segmentação de Drosophila / Interaction between the transcription factor CG9571 and cis-regulatory modules of the pair-rule gene even-skipped of Drosophila segmentation cascade

Gueller, Geison Castro da Silveira 22 May 2019 (has links)
O desenvolvimento do padrão de segmentação de Drosophila é estabelecido por uma cascata de genes de segmentação zigóticos. Estes genes são divididos em três classes (gap, pair-rule e segment-polarity) e codificam para fatores de transcrição (FT) que se ligam a módulos cis-reguladores (CRMs), reprimindo ou ativando genes alvo. A faixa 2 do gene pair-rule even-skipped (eve 2) é ativada pelos fatores maternos Bicoid e Hunchback e reprimida pelas proteínas gap Giant (Gt) e Krüppel. Estudos posteriores mostraram que o FT Sloppy-paired 1 (Slp 1) e provavelmente um outro FT forkhead também atuam na repressão de eve 2. O gene anotado CG9571 foi isolado em uma varredura como proteína forkhead candidata a repressão de eve 2. Estudos genéticos confirmaram essa possibilidade e revelaram que eve 1 também pode ser alvo deste FT. Este trabalho teve como objetivo verificar a interação de CG9571 com os CRMs eve 1 e eve 2. Para tanto, planejamos obter o domínio de ligação da proteína (CG9571 BD) e da proteína completa (CG9571 FL) e testar suas interações in vitro com fragmentos dos CRMs por meio da técnica de retardo da mobilidade eletroforética (EMSA). Obtivemos a quantidade necessária de DNA para os experimentos através de PCR e preparações plasmidiais de versões clonadas destes CRMs que já dispúnhamos em laboratório. Realizamos tentativas de obtenção de CG9571 BD por transcrição e tradução in vitro, mas esta estratégia não foi bem-sucedida e adotamos a estratégia de clonagem em vetor para expressão em células competentes bacterianas. O fragmento de CG9571 BD foi clonado com sucesso, mas não conseguimos verificar a expressão do polipeptídeo em duas linhagens de E. coli. Alteramos novamente nossa estratégia e clonamos o fragmento correspondente a CG9571 FL em vetor de expressão e conseguimos induzir sua expressão em bactéria, embora não tenha sido obter a proteína recombinante em forma solúvel. Prosseguimos para tentativas de recuperação da proteína a partir de corpos de inclusão. Foram empregados diferentes métodos para solubilização, renovelamento e purificação da proteína. Extratos da fração insolúvel solubilizada em diferentes concentrações de ureia foram submetidos a tentativas de purificação e renaturação por cromatografia de afinidade, mas não houve adsorção significativa de CG9571 FL em colunas com Ni2+ imobilizado. Preparações não puras contendo CG9571 FL foram obtidas através de procedimentos de renaturação destes extratos e foram utilizadas em ensaios de interação com os CRMs. Não houve detecção de retardo da mobilidade eletroforética dos fragmentos em gel. Foram observados efeitos de redução da quantidade de DNA detectado com brometo de etídio nas interações, mas este efeito foi considerado produto da ação de possíveis nucleases contaminantes nas preparações após investigação. Preparações de CG9571 FL puras foram obtidas por purificações a partir de SDS-PAGE, mas a maioria das interações da proteína solúvel com eve 1 e eve 2 não indicou formação de complexo. Obtivemos um único resultado positivo para a interação entre CG9571 FL e eve 2. Por não ter sido reproduzido, consideramos o resultado inconclusivo e novos experimentos serão conduzidos para dar continuidade à investigação da hipótese do trabalho / The development of Drosophila segmentation pattern is established by a cascade of zygotic segmentation genes. The zygotic genes are grouped in three classes (gap, pair-rule and segment-polarity) and code for transcription factors (TF) that bind to cis-regulatory modules (CRMs) with activation or repression roles. The stripe 2 of the pair-rule gene even-skipped (eve 2) is activated by the maternal factors Bicoid and Hunchback and repressed by the gap proteins Giant and Krüppel. Later studies showed that Sloppy-paired 1 (Slp 1) and probably another forkhead transcription factor also act for eve 2 repression. The annotated gene CG9571 was isolated in a search for putative forkhead protein repressors of eve 2. Genetic studies confirmed this possibility and reveled that eve 1 could also be a target for this TF. The aim of this work was to verify the interaction of CG9571 with the CRMs eve 1 and eve 2. To reach this aim, we planned to obtain the binding domain of the protein (CG9571 BD) or of the full-length protein (CG9571 FL) and to test their in vitro interactions with the eve 1 and eve 2 fragments by the electrophoretic mobility shift assay (EMSA). We obtained the necessary amount of DNA for the tests by PCR and plasmidial preparations of cloned versions of these CRMs that we already had in our laboratory. We made attempts to obtain CG9571 BD by in vitro transcription and translation system, but this strategy did not work and we adopted the cloning strategy to obtain the protein expressed by bacterial competent cells. CG9571 BD was cloned successfully, but we were not able to detect the polypeptide expression in two E. coli strains. We then turned to the CG9571 FL protein that we cloned and succeed to express it in bacteria, although we were not able to obtain the soluble recombinant form. We proceed for attempts of protein recovering from inclusion bodies. Different methods for solubilization, refolding and purification of the proteins were used. Extracts of the insoluble fraction solubilized in solutions with different urea concentrations were used in attempts of refolding and purification by affinity chromatography, but there was not significant CG9571 FL adsorption on columns with Ni2+ immobilized. We obtained impure preparations with CG9571 FL through procedures of refolding of these extracts and employed them on binding assays with the CRMs, but there was no gel shift detection. We observed reduction of the amount of DNA present in the binding reaction samples detected by ethidium bromide, but after further investigations this effect was considered the product of contaminant nuclease reaction from bacteria. Pure preparations of CG9571 FL were obtained by purification from SDS-PAGE, but there was no indication of complex formation on the most binding reaction assays with eve 1 and eve 2. We obtained only one positive result for the interaction between CG9571 FL and eve 2. However, the result was considered inconclusive because we were not able to reproduce it and new experiments will be conducted to investigate the hypothesis of this work
8

Mécanismes moléculaires de la transformation génétique naturelle chez la bactérie pathogène Helicobacter pylori / Molecular mechanisms of horizontal gene transfer in pathogen Helicobacter pylori

Celma, Louisa 03 April 2019 (has links)
Helicobacter pylori est une bactérie à Gram-négatif qui colonise la muqueuse de l’estomac humain. Elle se distingue des autres bactéries par un nombre de gènes très limité et de nombreuses particularités physiologiques et biochimiques. Elle provoque des infections associées à différentes maladies gastro-duodénales (ulcères et cancers). Depuis quelques années, une recrudescence de multi-résistances aux antibiotiques est observée. La transformation naturelle est l’un des processus clés qui les propage. Il s’agit d’un mécanisme de transfert horizontal de gènes qui permet aux bactéries de s’adapter à leur environnement, en internalisant des fragments d’ADN exogène à travers leur membrane, puis en les intégrant dans le chromosome par recombinaison homologue. Mes travaux ont visé à étudier de façon structurale et fonctionnelle trois protéines d’H. pylori décrites comme étant essentielles dans le processus de transformation naturelle: NucT, DprA et ComFc. La première partie de ce travail s’est concentrée sur la nucléase périplasmique NucT, supposée être impliquée dans la transformation chez H. pylori. Cependant, la délétion de son gène a permis de démontrer qu’elle ne joue en fait qu’un rôle mineur dans ce processus. La résolution de sa structure 3D a permis de mieux comprendre sa spécificité pour les acides nucléiques simple brin. Dans la seconde partie, la protéine DprA, responsable du chargement de la recombinase RecA sur l’ADN internalisé, a été étudiée. DprA d’H. pylori n’est composée que de 2 des 3 domaines qui constituent habituellement DprA, et fixe aussi bien l’ADN double brin que l’ADN simple brin mais uniquement via son domaine RF. Malgré son homologie structurale avec le domaine WH de liaison à l’ADN, le domaine C-terminal de HpDprA n’a pas d’affinité pour l’ADN. Nous avons mis en évidence des acides aminés conservés dans ce domaine dont l’étude pourrait permettre de comprendre son rôle. Enfin, une étude structurale de la protéine ComFc dont la délétion du gène entraîne la disparition totale de la capacité de transformation d’H. pylori a été réalisée. L’obtention de sa structure 3D a permis de mettre en évidence la présence d’un domaine catalytique phosphoribosyl-transférase ainsi que d’un domaine en doigt en zinc. Ce dernier pourrait être responsable de la capacité de ComFc à fixer l’ADN. Le substrat naturel de cette enzyme reste à découvrir.L’ensemble de ce travail a permis de contribuer à une meilleure compréhension à l’échelle moléculaire du mécanisme de transformation génétique naturelle d’H. pylori. L’avancement sur ces connaissances pourrait à long terme aider à réduire la propagation des multi-résistances par l’élaboration de nouvelles thérapies.Mots-clés : H. pylori, transformation naturelle, NucT, DprA, ComFc, interaction protéine-ADN / Helicobacter pylori is a Gram-negative bacterium that colonizes the mucus of the human stomach. It is distinguished from other bacteria by a limited number of genes and many physiological and biochemical characteristics. It causes infections associated with various gastro-duodenal diseases (ulcers and gastric cancers). In recent years, an increase in multi-resistance to antibiotics has been observed. Natural transformation is one of the key processes that spreads these multi-resistances. It is a horizontal gene transfer mechanism that allows bacteria to adapt to their environment by internalizing exogenous DNA fragments through their membrane and then integrating them into the chromosome by homologous recombination. My work aimed to study in a structural and functional approach three proteins of H. pylori described as essential in the natural transformation process: NucT, DprA and ComFc. The first part of this work focused on periplasmic nuclease, NucT, which is supposed to be involved in transformation in H. pylori. However, the deletion of its gene has shown that it actually plays only a minor role in this process. The resolution of its 3D structure has led to a better understanding of its specificity for single-stranded nucleic acids. In the second part, the protein DprA, responsible for loading RecA recombinase onto internalized DNA, was studied. HpDprA is composed of only 2 of the 3 domains that usually constitute DprA, and binds both double-stranded and single-stranded DNA but only via its RF domain. Despite its structural homology with the WH DNA binding domain, the C-terminal domain of HpDprA has no affinity for DNA. We have identified conserved amino acids in this domain that could be studied to understand its role. Finally, a structural study of ComFc, whose deletion of the gene leads to the total disruption of the transformation capacity of H. pylori, has been carried out. The acquisition of its 3D structure has highlighted the presence of a phosphoribosyl transferase catalytic domain as well as a zinc finger domain. The latter could be responsible for capacity of ComFc to bind DNA. The natural substrate of this enzyme remains to be discovered.All this work has contributed to a better knowledge at the molecular level of the natural genetic transformation mechanism of H. pylori. Advancing this knowledge could in the long term help to reduce the spread of multiresistance through the development of new therapies.Keywords: Helicobacter pylori, natural transformation, NucT, DprA, ComFc, protein-DNA interaction
9

Molekulární mechanismy rozpoznání regulačního úseku DNA transkripčními faktory z rodiny MADS-BOXU / Molecular mechanism of DNA regulatory segment recognition by MADS box family transcription factors

Profantová, Barbora January 2014 (has links)
The thesis deals with physico-chemical properties of the MADS box, binding domain of transcription factors, which are important for the formation of complexes with the DNA regulatory segment bearing the CArG box. The study was performed also on model oligopeptides, selected segments of the MADS box and their analogues with a point mutation. A wide range of spectroscopic techniques was employed, namely absorption, circular dichroism, fluorescence and Raman spectroscopies. Advanced approaches including multivariate methods were used for data processing. The three tyrosines of the MADS box located in amino-acid vicinities of different charge and hydrophobicity, were used as intrinsic spectroscopic probes. The obtained characteristics of the MADS box and its segments structural arrangement, flexibility and acid-base equilibria are the main results of the work.
10

Interaction of bZIP and bHLH Transcription Factors with the G-box

De Jong, Antonia Thelma-Jean 07 August 2013 (has links)
Transcription factors are proteins that regulate transcription of genes by binding to specific DNA sequences proximal to the gene. The specificity and affinity of protein-DNA recognition is critical for proper gene regulation. This thesis explores the mechanisms of binding to the sequence 5’CACGTG, a common recognition sequence both in plants where it is known as the G-box and in mammalian cells where it is termed the E-box. This sequence is of clinical interest because it is the target of the transcription factor Myc, an oncogene linked to many cancers. A number of alpha-helical proteins with different dimerization elements, from the basic region-leucine zipper (bZIP), basic region helix-loop-helix leucine zipper (bHLHZ) and basic region helix-loop-helix-PAS (bHLH-PAS) protein families, are capable of binding to this sequence. The basic regions of all these protein families contain residues that contact DNA and determine DNA sequence specificity while the other subdomains are responsible for dimerization specificity. First, the influence of protein-DNA contacts on sequence specificity of the plant bZIP protein EmBP-1 was probed by point mutations in the basic region. Residues that contact the DNA outside the core G-box sequence and residues that contact the phosphate backbone were found to be important for sequence specificity. Second, the impact of the dimerization subdomains of bHLHZ protein Max, the required heterodimerization partner of the Myc protein, and bHLH-PAS protein Arnt was probed by mutation, deletion and inter-family subdomain swapping studies. All studied protein families are intrinsically disordered, forming structure upon dimerization and DNA binding. The dimerization domains were found to indirectly influence DNA binding by affecting folding, dimerization ability or proper orientation of the basic regions relative to DNA. Lastly, a new strategy for selection of G-box binding proteins in the Yeast One-hybrid system is explored. Together, these studies broaden our understanding of the structure-function relationship of the DNA-binding activities of these closely related families of transcription factors. The creation and characterization of mutants with altered specificity, affinity and dimerization specificity may also be useful for biotechnology applications.

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