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Vnitrobuněčná a mezibuněčná regulace genové exprese u Gram-pozitivních bakterií / Intracellular and intercellular regulation of gene expression in Gram-positive bacteria.Pospíšil, Jiří January 2021 (has links)
Bacteria, the most dominant organisms on Earth, are an everyday presence in our lives. Symbiotic bacteria, which are present in the digestive tract of animals, usually have a beneficial effect on the body. On the opposite side of the spectrum are pathogenic species that cause more or less serious diseases around the world. In order to fight pathogens effectively, it is necessary to learn as much as possible about the molecular mechanisms by which bacteria respond to their environment, and also about the types of communication within bacterial populations that allow them to react to environmental changes as "multicellular" organisms. This Thesis consists of two main parts. In the first part, selected aspects of bacterial gene expression are characterized, using Bacillus subtilis and Mycobacterium smegmatis as model organisms. DNA-dependent RNA polymerase (RNAP) is the enzyme that is responsible for transcription of DNA into RNA, and thus plays a key role in gene expression. This Thesis deals with the structure of bacterial RNAP and important auxiliary factors (proteins and RNA) that associate with this enzyme and modulate its function. In the second part, the focus is on cell-to-cell communication, revealing which factors/mechanisms, including gene expression, affect this process in B. subtilis....
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Detecting RNA Regulatory Interactions in Bacterial CellsKuryllo, Kacper 11 1900 (has links)
Non-coding RNAs are involved in the regulation of most major cellular process in Escherichia coli. With current technologies, many of these molecules have been identified; however, the full scope of their regulatory interactions is still unknown. None of the techniques currently in use employ the regulatory effect of the RNAs, which is the major unifying attribute of these molecules, for their identification. This thesis presents projects involving the design of a dual-reporter plasmid and screening method in the discovery and characterization of RNA regulatory interactions
The first project details the engineering of the dual reporter plasmid. This vector utilizes one fluorescent protein to detect regulatory events and a second to normalize for off-target effects. The second project utilizes this tool in the discovery and characterization of novel regulatory responses. This is accomplished by screening a library of intergenic regions for regulatory responses against a collection of metabolite. Interesting interactions involving nitrogen abundance, iron and uracil are identified and further examined.
Finally, this thesis examines how this technology can be further expanded for the study of RNA regulatory functions. The use of the screening method for the detection of regulatory events caused by alternative minimal media composition and the potential for the dual reporter plasmid to aid in the study of riboswitches are investigated. / Thesis / Doctor of Science (PhD)
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<b>Role of MicroRNA in Canine Diffuse Large B-Cell Lymphoma</b>Nelly O Elshafie IV (17104207) 06 October 2023 (has links)
<p dir="ltr">Lymphoma is a prevalent malignancy in dogs. Diffuse large B-cell Lymphoma (DLBCL) is the common subtype that represents about 50% of the clinically seen lymphoma cases. DLBCL diagnosis relies on cytological examination of a fine needle aspirate and histological evaluation by immunohistochemistry (IHC) in most common practices. This workflow is sufficient to confirm the diagnosis; however, it may be challenging to differentiate reactive and neoplastic forms in some controversial cases. In such cases, PCR-based clonality assays and flow cytometry (FC) can help with more conclusive diagnoses. So, finding more biomarkers that can detect and track DLBCL early and over time is a must for a final diagnosis and helps us learn more about how DLBCL starts at the molecular level. MicroRNAs (miRNAs), the small non-coding RNAs, regulate gene expression by binding to the 3'-untranslated region of protein-coding RNAs, leading to either RNA degradation or translational repression. They can switch on and off genes to regulate physiological and pathological processes. MicroRNA stability features and tissue availability make them promising biomarkers for identifying and sub-classifying patients and sequentially evaluating the disease status or the response toward a specific medicine. This dissertation investigates the small RNA sequence analysis, the differentially expressed miRNAs between healthy and DLBCL-affected lymph nodes, and the miRNA profile in DLBCL cases with different outcomes.</p>
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Identification of a Fur-regulated small regulatory RNA in nontypeable <i>Haemophilus influenzae</i>Santana, Estevan Alexis January 2014 (has links)
No description available.
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Developing Molecular Tools for Applications in Metabolic Engineering and ProteinPurificationlahiry, ashwin January 2017 (has links)
No description available.
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Small RNA Sibling Pairs RyfA and RyfB in <i>Shigella dysenteriae</i> and their Impact on PathogenesisFris, Elizabeth Megan 01 October 2018 (has links)
No description available.
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Characterization of SraB, a novel small RNA molecule, in the pathogenic bacterium Shigella dysenteriaeAlmasude, Eden 16 June 2011 (has links)
No description available.
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Stochastic Modeling of Gene Expression and Post-transcriptional RegulationJia, Tao 19 August 2011 (has links)
Stochasticity is a ubiquitous feature of cellular processes such as gene expression that can give rise to phenotypic differences for genetically identical cells. Understanding how the underlying biochemical reactions give rise to variations in mRNA/protein levels is thus of fundamental importance to diverse cellular processes. Recent technological developments have enabled single-cell measurements of cellular macromolecules which can shed new light on processes underlying gene expression. Correspondingly, there is a need for the development of theoretical tools to quantitatively model stochastic gene expression and its consequences for cellular processes.
In this dissertation, we address this need by developing general stochastic models of gene expression. By mapping the system to models analyzed in queueing theory, we derive analytical expressions for the noise in steady-state protein distributions. Furthermore, given that the underlying processes are intrinsically stochastic, cellular regulation must be designed to control the`noise' in order to adapt and respond to changing environments. Another focus of this dissertation is to develop and analyze stochastic models of post-transcription regulation. The analytical solutions of the models proposed provide insight into the effects of different mechanisms of regulation and the role of small
RNAs in fine-tunning the noise in gene expression. The results derived can serve as building blocks for future studies focusing on regulation of stochastic gene expression. / Ph. D.
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Regulatory RNAs in Staphylococcus aureus : Function and Mechanism / ARN régulateurs chez Staphylococcus aureus : Fonction et MécanismeLe Lam, Thao Nguyen 24 September 2015 (has links)
Le staphylocoque doré (S. aureus) est un pathogène responsable d’infections diverses chez l’homme et l’animal. Il est responsable d’infections suppuratives (furoncles, endocardites, méningites…) ou non suppuratives (toxi-infections alimentaires…). L’émergence de souches bactériennes multi-résistantes aux antibiotiques augmente la gravité des infections et constitue un réel problème de santé publique. Depuis plusieurs années, les chercheurs tentent d’identifier des cibles pour de nouveaux antibiotiques. Parmi elles, les acides ribonucléiques (ARN) dits « régulateurs » constituent un modèle de choix. Environ 200 ARN régulateurs ont été identifiés chez S. aureus, mais jusqu'à maintenant, dans la plupart des cas, leurs fonctions ne sont pas connues. Pour identifier la fonction des ARN régulateurs chez S. aureus, nous avons utilisé une stratégie consistant à mettre en compétition 39 souches différentes dans chacune desquelles un ARN régulateur a été remplacé par une étiquette spécifique identifiable par séquençage. Cette expérience de compétition a été réalisée dans douze conditions de cultures différentes (conditions de stress différents) ainsi que chez un modèle souris. Les résultats de ces expériences ont été obtenus par analyse de séquençage massif haut débit. Un phénotype a été mis en évidence pour 11 des ARN régulateurs étudiés dans les conditions choisies. Nous avons aussi développé une méthode fiable pour identifier les cibles des ARN régulateurs. Cette méthode consistait à utiliser in vitro, ces ARN régulateurs comme appât pour piéger leurs cibles parmi un mélange d’ARN total. Les ARN cibles piégés ont ensuite été séquencés par ARN-seq haut débit. Cette stratégie a été appliquée pour déterminer les cibles de quatre ARN régulateurs chez S. aureus : RsaA, RsaE, RsaH et RNAIII. Plusieurs cibles putatives ont été identifiées et utilisées pour prédire le motif qui serait impliqué dans les interactions entre un ARN régulateur et sa cible. Le dernier chapitre de ce manuscrit concerne l’étude du mécanisme d’action des ARN régulateurs. En général, la liaison de l’ARN régulateur à son ARN messager cible va affecter la stabilité de ce dernier et engendrer sa dégradation par la ribonucléase III (RNase III). Chez S. aureus, RNase III est l’enzyme principalement impliquée dans la dégradation des complexes ARN régulateur-ARN messager (ARN double brin). RNase III a été décrite comme étant non essentielle chez S. aureus. Cependant, nous avons montré que cette ribonucléase est essentielle dans les souches de S. aureus contenant des prophages portant un système toxine/antitoxine (TA) de type I. Dans ces souches, la présence de RNase III est essentielle pour diminuer l’expression de ce système TA et contrer sa toxicité. / Staphylococcus aureus is an opportunistic Gram-positive pathogen bacterium and a leading cause of hospital- and community-acquired infections worldwide. In S. aureus, regulatory RNAs are key mediators in controlling bacterial virulence, viability and adaptability under various environmental conditions. About 200 regulatory RNAs were identified in S. aureus but up to date, their functions in most cases are unknown. To address the function of S. aureus regulatory RNAs, we used a strategy in which competitive fitness experiments were performed with mixed cultures comprising thirty-nine sRNA mutants. A bacterial population made of these mixed mutants was grown in twelve stress conditions and in a mouse model. The results were obtained by deep sequencing analysis. Eleven sRNA gene mutants were found to have an altered fitness in the tested conditions; three of them being requires for solely one studied condition, the others being modified by multiple stress conditions. The absence of sRNA genes generated negative fitness adaptation in all conditions, but one of the mutants had a strong growth defective phenotype at low-temperature. Current investigations indicate that the deleted sequence affects the 3’ end of a mRNA. This sequence is required for an optimum growth in cold conditions and contributes to the stability or translation of the associated mRNA. We also developed a robust procedure to identify reliably sRNA targets based on synthetic sRNAs that were used in vitro as bait to trap their corresponding targets which were subsequently identified by deep sequencing. Using this approach, we found new targets for RsaA, RsaE, RsaH and RNAIII. The binding of sRNAs to targeted mRNAs usually affect their stability by recruiting Ribonucleases (RNases). In S. aureus, RNase III encoded by rnc gene, is a major RNase involved in the degradation of sRNA-mRNA duplexes. RNase III was reported as nonessential in S. aureus. However, we report that the rnc gene is essential in some S. aureus strains due to the presence of prophages carrying type I toxin/antitoxin (TA) system. RNase III in these strains is essential to reduce the expression of TA systems to prevent their toxicity
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Virulence et résistance aux antibiotiques du staphylocoque doré : recherche des ARNm ciblés par deux ARN régulateurs / Staphylococcal virulence and antibiotic resistance : research of mRNA targeted by two sRNAsIvain, Lorraine 12 July 2017 (has links)
Staphylococcus aureus est une bactérie pathogène opportuniste de l’Homme et de l’animal qui représente l’une des premières causes d’infections nosocomiales. A l’heure actuelle, la sévérité de ces infections est accentuée par l’augmentation des multi-résistances aux antibiotiques qui en font un problème majeur de santé public. La découverte et l’étude des ARN régulateurs (ARNrég) exprimés par le staphylocoque doré a permis de mettre en évidence leurs rôles de régulateurs de l’étape de colonisation et d’infection (quorum sensing, virulence, résistance aux antibiotique, adaptation environnementales). Au cours de ma thèse, je me suis intéressée à l’étude de deux ARNrég, SprD impliqué dans la virulence de la bactérie sur modèle animal murin et SprX régulant la résistance aux glycopeptides. Dans le but de comprendre les bases moléculaires de leurs rôles dans la bactérie, nous avons souhaité identifier et valider l’ensemble des cibles directes à ces deux ARN. Dans un premier temps, nous nous sommes intéressés à l’étude des nouvelles cibles de SprX. Pour cela, nous avons mis en place une approche in vivo de validation des cibles des ARNrég basée sur la technique des gènes rapporteurs. Nous avons ainsi pu valider deux nouvelles cibles de SprX, les ARNm ecb et purC impliqués respectivement dans l’évasion du système immunitaire et dans le métabolisme des purines. SprX interagit au niveau du site d’initiation de la traduction de ces deux ARNm, et inhibe leurs traductions. Par la suite, nous avons souhaité rechercher de nouvelles cibles pour SprD. Nous avons pu adapter à S. aureus une technique d’identification de nouvelles cibles des ARNrég. L’ensemble des résultats obtenus permettent de préciser les rôles déjà connus des deux ARNrég et offrent de nouvelles possibilités concernant l’étude et la validation des cibles potentielles. / Staphylococcus aureus is an opportunistic pathogen that causes a wide variety of nosocomial and community-acquired infections. Its adaptive capacities, multiple antibiotic resistances and high virulence makes it a major public health issue. The discovery and study of small regulatory RNAs (sRNAs) expressed by S. aureus showed their implication in the switch colonization to host infection. During my phD, I focused on the study of two sRNAs: (i) SprD, involved in staphylococcal virulence and (ii) SprX which regulates glycopeptides resistance. To understand the molecular basis of their roles in staphylococcal physiology, we searched for their direct targets using two different techniques. For SprX, we set up an in vivo technique to validate mRNA targets previously predicted using bioinformatics tools. This method based on the use of a fluorescent reporter gene allowed us to validate two novels SprX targets, the ecb and purC mRNAs involved in immune evasion system and purine metabolism, respectively. We further showed that SprX interacts at the ribosome binding sites preventing translation initiation. In another chapter of my phD project, we adapted the MAPS technique to S. aureus and applied it to the search of SprD direct partners by affinity purification of sRNA-mRNA duplexes. Overall, the results obtained during my thesis allowed us to precise the roles of SprD and SprX and will offer novel possibilities for the staphylococcal community to identify and/or validate of sRNA targets.
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