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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.
51

Transcriptional crosstalk between helper bacteriophages and Staphylococcal aureus pathogenicity islands

Lane, Kristin 05 December 2013 (has links)
Acquisition of a superantigen pathogenicity island (SaPI) significantly increases virulence in Staphylococcus aureus. Horizontal transfer of SaPIs occurs at high frequency and depends upon a helper bacteriophage, either through direct infection or SOS-mediated induction of a lysogen. SaPIs hijack the packaging machinery of the helper phage, leading to the formation of SaPI-containing transducing particles that can introduce the pathogenicity island into neighboring SaPI-negative cells. All SaPIs contain a conserved core of genes, some of which are co-transcribed as an operon and encode functions involved in helper exploitation. The goal of this study was to more fully understand the intricate relationships between the SaPI elements and their helper bacteriophages, specifically any regulatory crosstalk that might occur between them. We demonstrated phage-host interactions in 80 and 80α, and SaPI1 and SaPIbov1-mediated crosstalk with helper phage 80α. The phage Sri protein was shown to be a bi-functional protein that both derepresses SaPI1 and interferes with host chromosome replication. Incoming SaPI1 experiments showed that SaPI1 modulates the levels of the N-terminal part of orf14 mRNA. Induction experiments using the 80α ΔrinA phage as a genetic tool, reveal several new phage genes that SaPI1 targets for expression modulation. Finally, a novel SaPI1 interference mechanism was identified. In an 80α ΔrinA mutant, which cannot activate its late operon, SaPI1 can directly turn on expression of the packaging and structural genes in a noncanonical manner, initiating from the 2nd gene in the operon, the large terminase subunit.
52

Physiological and Molecular Characterization of Genetic Competence in Streptococcus sanguinis

Rodriguez, Alejandro 21 July 2008 (has links)
The ability of bacteria to assimilate free DNA from the environment is known as competence. Though many studies have focused on competence regulation in Streptococcus pneumoniae and Streptococcus gordonii, Streptococcus sanguinis has yet to be examined. Physiological characterization of competence in S. sanguinis strain SK36 and its comC mutant, JFP41, led to the genome-wide transcriptional analysis of cells induced to competence via addition of competence-stimulating peptide (CSP). A total of 128 genes were induced at least 2-fold, 74 of which were classified as either “early” or “late” based on their induction patterns. Expression patterns were verified using qRT-PCR. This study identified genes not up-regulated in S. pneumoniae or S. gordonii and lays the foundation for bioinformatic studies to identify conserved binding sites upstream from CSP-regulated genes. These results also shed light on the possible existence and identity of expected CSP exporters in S. sanguinis, which have so far eluded discovery.
53

Reportérový expresní systém pro studium umlčování integrovaného proviru v transkribované oblasti genu / Reporter expression system for study of silencing of provirus integrated inside transcriptionally active gene

Slavková, Martina January 2015 (has links)
Retroviral vectors are used as mighty tools for an introduction of recombinant genes into the recipient genome in gene therapy trials. In the vector design, great emphasis is put on safety and efficiency. In spite of a great progress in retroviral vector design with the purpose to stabilize its expression, e.g. introduction of protective elements into the viral regulatory sequences, the current approaches are still not sufficiently effective and the majority of proviruses is transcriptionally silenced. The understanding of the silencing mechanism is of special importance to the optimization of the vector design and handling. In this master thesis, I have designed and constructed an expression system for study of the mechanism involved in the silencing of retroviruses integrated inside gene bodies. This artificial system will be utilized for testing of hypothesis that retroviruses integrated into gene bodies are silenced by DNMT-dependent mechanism and this process is triggered by transcriptional read-through of the provirus from nearby host promoter. I have obtained preliminary results suggesting the validity of the suggested hypothesis; however the verification of general validity of this hypothesis for various retroviruses and elements will be a matter of further studies in our laboratory. Powered by...
54

Engineering virus resistant transgenic cassava: the design of long hairpin RNA constructs against South African cassava mosaic virus

Harmse, Johan 19 March 2008 (has links)
ABSTRACT Cassava is currently the second most important source of carbohydrates on the African continent. In the last two decades, cassava crops have been severely affected by outbreaks of cassava mosaic disease (CMD). South African cassava mosaic virus (SACMV) has been associated with CMD outbreaks in the Mpumalanga province. Advances in post-transcriptional gene silencing (PTGS) technology have provided promising new strategies for the engineering of virus resistance in plants. Inverted repeat (IR) constructs are currently the most potent inducers of PTGS, however, these constructs are inherently unstable. The purpose of this study was to develop IR constructs with an improved stability for the efficient induction of PTGS in plants. Two mismatched inverted repeat constructs, one targeting the SACMV BC1 open reading frame, the other targeting the Maize streak virus (MSV) AC1 open reading frame, were successfully created. Sodium bisulfite was used to deaminate cytosine residues on the sense arm of the constructs. The resulting number of GT mismatches was seemingly sufficient to stabilize the linear conformation of the IR constructs, as they were efficiently propagated by E.coli DH5!, and subsequently behaved like linear DNA molecules. Furthermore, it was found that the number of mismatches on the BC1 construct (17.5%) was ideal, as the subsequent stability of the predicted RNA hairpin was not affected. Due to the higher number of mismatches on the AC1 construct (23.5%), it was found that the loop region of the RNA hairpin was marginally destabilized. Despite this, long stretches of stable dsRNA were still produced from the AC1 IR construct, and is likely to induce PTGS. Interestingly, it was observed that the mismatched IR constructs, although still replicated in E.coli, were marginally destabilized in Agrobacterium. Therefore, it was deduced that the stability of a mismatched IR construct may be influenced by the particular intracellular environment of an organism. Due to the recalcitrance of cassava to transformation, a model plant system, Nicotiana benthamiana, was used to screen constructs for toxicity, stability, and efficiency of PTGS induction. Agrobacteriummediated transformation and regeneration of N. benthamiana was optimized, and 86% transformation efficiency was achieved when using leaf disk explants. It was found that the addition of an ethylene scrubber, potassium permanganate, substantially increased the rate of regeneration by reducing the frequency of hyperhydritic plants. Transgene iv integration was confirmed by PCR amplification of the hptII gene in the T-DNA region. Transgene expression was confirmed by screening for GUS and GFP reporter genes. No toxic responses to the transgene have been observed thus far. Studies are currently underway to confirm the stability of the mismatched IR constructs in N. benthamiana. PAGE Northern blotting is being done, as the detection of siRNAs derived from the transgene will confirm that constructs are functional. In addition, infectivity assays are underway to determine the efficacy of BC1 knockdown by a stably integrated construct. Due to the enhanced stability of mismatched IR constructs, they may be an appealing alternative to currently available intron-spliced, or exact matched hairpin systems.
55

Expressão e purificação da quinase dependente de ciclina 13 humana em sistema bacteriano / Expression and purification of human cyclin-dependent kinase 13 in bacterial system

Moreira, Juliana 10 April 2014 (has links)
As quinases dependentes de ciclinas são proteínas que podem ser divididas de acordo com a sua atuação no ciclo celular ou no controle transcricional, elas se tornam ativas em determinadas etapas do ciclo celular dependendo do seu grau de fosforilação e de sua ligação com ciclinas e proteínas inibitórias, e exercem sua função fosforilando outras proteínas envolvidas no ciclo de divisão celular e transcrição influenciando suas atividades, garantindo que cada processo do ciclo ocorra em uma sequência ordenada. A CDK13 faz parte da família de proteínas quinases dependentes de ciclina, pode se ligar a ciclinas do tipo L ou K, regula os eventos de \"splicing\" alternativo, e interage com a proteína Tat do vírus HIV atuando como um possível fator de restrição, sendo que sua superexpressão diminui a produção de algumas proteínas virais suprimindo a produção do vírus. O DNA referente à CDK13 é replicado em células cancerosas, principalmente dos tipos hepático e cólon e reto, sendo um alvo para inibidores para tratamento de câncer. A fim de contribuir para o estudo dessa proteína, o projeto tem como objetivo expressá-la utilizando métodos de tecnologia de DNA recombinante. A sequência de DNA referente à CDK13 foi amplificada pela reação em cadeia da polimerase, após sua purificação, foi inserida no vetor pCR-Blunt e clonada em células de E. coli DH5α competentes. Porém, o DNA não foi liberado pela reação com as enzimas de restrição BamHI e NdeI. As bactérias Rosetta(DE3) transformadas com um plasmídeo sintético e crescidas em meio de auto-indução expressaram a CDK13. Após lise celular e purificação em coluna de Ni2+, a proteína foi detectada por Western Blot. Já as bactérias Rosetta(DE3) transformadas com o plasmídeo sintético modificado (o qual compreende a região do DNA que expressa o bolsão de ligação da CDK13), e induzidas em meio LB expressaram a CDK13, porém não foi possível purificá-la em coluna de afinidade ao Ni2+. / The cyclin-dependent kinases are proteins that can be classified by their function in the cell cycle or transcriptional control. They are activated in particular steps of the cell cycle depending on their phosphorylation degree, cyclin binding and inhibitory proteins. They act phosphorylating other proteins involved in the cell cycle and transcriptional control, influencing in their activities, ensuring that each step of the cell cycle occur in an ordered sequence. The CDK13 is one of the cyclin-dependent kinases family member, it can bind to L or K cyclins, regulates the alternative splicing and interact with HIV Tat protein, acting as a possible restriction factor, its overexpression decreases the production of some viral proteins, and suppresses the virus production. The DNA corresponding to CDK13 is replicated in cancer cells, mainly of hepatic and colon rectal types; therefore it is a target for inhibitors for cancer therapy. In order to contribute for the studies of this protein, the goal of the project is to express it using methods of recombinant DNA technology. The DNA sequence corresponding to CDK13 was amplified by polymerase chain reaction, after its purification, it was inserted to pCR-Blunt vector and cloned into E. coli DH5α competent cells. However, the DNA wasn\'t released by the BamHI and NdeI restriction enzymes. The Rosetta(DE3) cells transformed with a synthetic plasmid pET28a::CDK13 and grown in auto-induction media expressed the CDK13. After cell lysis and purification by Ni2+ affinity colum, the protein was identified by Western Blot. However, the Rosetta(DE3) cells transformed with the modified synthetic plasmid (that comprehends the DNA region which expresses the binding pocket region) induced in LB media, expressed the CDK13. Yet, it wasn\'t possible to purify the protein in the Ni2+ affinity column.
56

Unveiling the effect of global regulators in the regulatory network for biofilm formation in Escherichia coli / Entendendo o efeito dos reguladores globais na rede regulatória para a formação de biofilme em Escherichia coli

Amores, Gerardo Ruiz 29 March 2017 (has links)
In nature, biofilm is a complex structure resulted of multicellular bacterial communities that provide important nutritional functions and the acquisition of protective traits such as antibiotics resistance and horizontal gene transfer. The development from the planktonic, lonely bacteria, to the mature multilayered biofilm structure consists of three main phases: motility, attachment and biofilm maturation. At cellular level, the process is controlled by several genes such as flhD, fliA, rpoS, csgD, adrA, cpxR all acting as master regulators. Additionally, the global regulators CRP, IHF, Fis, and others in less frequency, have been related to biofilm formation, although blurry information has been provided. In this thesis we used synthetic, molecular and cellular biology approaches to understand the effect of CRP, IHF and Fis in the transcriptional regulatory network in the bacterium Escherichia coli. In the first chapter, we employed network analysis to reconstruct and analyze part of the entire regulatory network described to modulate the flagella-biofilm program. With this analysis we identified some critical interactions responsible for the planktonic-biofilm transition. Next, we selected the top ten effectors nodes of the network and cloned the promoter region of those genes in a reporter system. As extensively explained in chapter II, this system allowed us to validate as well as suggest new interactions in the network. Additionally, the measurement of the promoter activity during bacterial development show that CRP, IHF and Fis differentially modulate most of the surveyed genes suggesting that those Global Regulators participate to modulate gene expression in different phases of the planktonic-biofilm development. At chapter three, to get a better overview of the entire process, we performed motility, adherence/early biofilm and mature biofilm assays. We describe the intrinsic ability of E. coli to perform motility, adherence and mature biofilm at 37?C. In contrast, the absence of ihf, fis as well as Carbon Catabolite Repression (CCR), lead to altered phenotypes at both motility and biofilm development. At the end, we discussed how the changes of promoter activity of target genes, together with our network analysis, could explain part of the altered phenotypes observed. For instance, we observed changes at the main stress responders rpoS and rpoE that, in combination with alterations at specific genes such as fliA, can explain the enhanced motility in the E. coli ?ihf strain. Altogether, in this thesis, we provided evidence that CRP, IHF and Fis control the activity of the promoter regions of genes involved in the planktonic-biofilm development. / Na natureza, o biofilme é uma estrutura complexa resultante de comunidades bacterianas multicelulares que fornece importantes funções nutricionais e a aquisição de traços de proteção como resistência a antibióticos e transferência horizontal de genes. O desenvolvimento das bactérias planctônicas solitárias para uma estrutura de biofilme maduro consiste em três fases principais: motilidade, fixação e maturação do biofilme. Ao nível celular, o processo é controlado por vários genes tais como flhD, fliA, rpoS, csgD, adrA, cpxR, todos agindo como reguladores mestre. Além disso, os reguladores globais CRP, IHF, Fis e outros em menor freqüência, têm sido relacionados à formação de biofilme, embora tenham sido fornecidas informações nao conclusivas sobre esse processo. Nesta tese foram utilizadas abordagens de bioinformática, assim como de biologia molecular e celular para entender o efeito de CRP, IHF e Fis na rede reguladora da transição de motilidade para biofilme na bactéria Escherichia coli. No primeiro capítulo, utilizamos a análise de rede para reconstruir e analisar parte da rede regulatória descrita para modular o programa flagelo-biofilme. Com esta análise identificamos algumas interações críticas responsáveis pela transição planctônica-biofilme. Em seguida, selecionamos os dez principais nós efetores da rede e clonamos a região promotora desses genes em um sistema repórter. Conforme explicado amplamente no capítulo II, este sistema nos permitiu validar e sugerir novas interações na rede. Adicionalmente, a medição da atividade do promotor durante o desenvolvimento bacteriano mostra que a CRP, a IHF e a Fis modulam diferencialmente a maioria dos genes analisados sugerindo que estes Reguladores Globais participam para modular a expressão génica em diferentes fases do desenvolvimento de estado planctónico para biofilme. No capítulo três, para obter uma melhor visão geral de todo o processo, realizamos ensaios de motilidade, aderência / biofilme precoce e biofilmes maduros. Descrevemos a capacidade intrínseca de E. coli para realizar motilidade, adesão e biofilme maduro a 37 °C. Em contraste, a ausência de ihf, fis, bem como o fenômeno de Repressão de Catabolite de Carbono (CCR), levam a fenótipos alterados, tanto na motilidade como no desenvolvimento do biofilme. No final, discutimos como as mudanças da atividade do promotor de genes alvo, juntamente com a nossa análise de rede, poderia !xi explicar parte dos fenótipos alterados observados. Por exemplo, observamos mudanças nos principais respondedores de estresse rpoS e rpoE que, em combinação com alterações em genes específicos como fliA, podem explicar a motilidade aumentada na estirpe de E. coli ?ihf. Em conjunto, nesta tese, apresentamos evidências de que CRP, IHF e Fis controlam a atividade das regiões promotoras de genes envolvidos no desenvolvimento planctônico-biofilme.
57

Elementos repetitivos na regulação da transcrição de Mycoplasma hyopneumoniae

Cattani, Amanda Malvessi January 2016 (has links)
Mycoplasma hyopneumoniae é uma bactéria de tamanho diminuto, caracterizada por um genoma pequeno, com baixo conteúdo GC. Está associada com doenças respiratórias de suínos, resultando em prejuízos produtivos e econômicos na indústria animal. A presença de sequências de DNA repetitivas, que ocorrem em grandes quantidades em células eucarióticas, vem sendo cada vez mais identificadas em genomas de procariotos, sendo também associadas a um potencial papel regulador. Uma vez que a regulação da transcrição nesses organismos ainda é pouco entendida, o objetivo do presente estudo foi realizar uma busca in silico por elementos repetitivos nas regiões intergênicas do genoma de M. hyopneumoniae linhagem 7448. Dois tipos de repetições foram selecionados para a busca inicial: tandem e palindromes. Regiões intergênicas de até 500 pb a montante do sítio de início da tradução de todas as CDSs do genoma de M. hyopneumoniae linhagem 7448 foram utilizadas para a predição. Para cada tipo de elemento dois programas computacionais independentes foram utilizados. As predições in silico resultaram em 144 repetições em tandem e 1.171 palindromes. O DNA repetitivo se encontra distribuído a montante de 86% das unidades transcricionais de M. hyopneumoniae linhagem 7448. Análises comparativas entre genomas de micoplasmas demonstraram diferentes níveis de conservação dos elementos repetitivos entre linhagens patogênicas e não-patogênicas. Linhagens patogênicas revelaram uma conservação de 59%, enquanto que a não patogênica, somente de 46%. Através de ensaios de amplificação quantitativa de DNA, foi observado diferentes níveis de expressão em genes codificantes para importantes proteínas, como glicina hidroximetiltransferase, lipoproteína, adesinas e proteína ligadora de GTP. Os genes codificantes para essas proteínas divergiam no número de repetições palindromes e tandens na sua respectiva região intergênica. Além disso, repetições encontradas em 206 genes já descritos como regulados em diferentes condições em M. hyopneumoniae linhagem 232 mostraram aproximadamente 80% de conservação em relação à linhagem M. hyopneumoniae linhagem 7448. Todos esses resultados sugerem um potencial papel regulador das repetições de DNA em tandem e palindromes em Mycoplasma. / Mycoplasma hyopneumoniae is a diminutive bacterium, characterized by a small genome with a low GC content. It is commonly associated with swine respiratory diseases, resulting in productivity and economic losses in the animal industry. Repetitive DNA, which occurs in large quantities in eukaryotic cells, has been increasingly identified in prokaryotic genomes, and has been associated with a potential regulatory function. Once transcription regulation in these organisms is still poorly understood, the aim of the current study was to perform an in silico search of repeat elements in the genomic intergenic regions of M. hyopneumoniae strain 7448. Two types of repeats were selected for initial search: Tandem and Palindromic. Intergenic regions up to 500 bp upstream from start codon of M. hyopneumoniae strain 7448 CDSs were used as input for the software’s prediction. For each type of repeat sequence, two independent software packages were used. Computational analysis results in 144 tandem repeats and 1,171 palindrome elements. The repeats were distributed in the upstream region of 86% of transcriptional units of M. hyopneumoniae strain 7448. Comparative analysis between distinct mycoplasmas, demonstrate different indices of repeat conservation among pathogenic and non-pathogenic strains. Pathogenic strains revealed 59% conservation, while non-pathogenic only 46%. Through assays of quantitative amplification of DNA, different levels of expression in genes coding important proteins have been demonstrated, as glycine hydroxymethyltransferase, lipoprotein, adhesins and GTP-binding protein. These protein coding genes differ in number of palindromes or tandem repeats in respective upstream regions. In addition, repeats found in 206 genes already described to be regulated in different grow conditions in M. hyopneumoniae strain 232 showed almost 80% of conservation in relation to M. hyopneumoniae strain 7448. All these findings, suggests a potential regulatory role of tandem and palindrome DNA repeats.
58

Caractérisation moléculaire et fonctionnelle de la protéine DYW1 dans le complexe d'édition chloroplastique d'Arabidopsis thaliana / Molecular and functional characterization of the DYW1 protein in the chloroplast editing complex of Arabidopsis thaliana

Boussardon, Clément 02 April 2013 (has links)
Dans les organites des plantes, l’édition de l’ARN consiste majoritairement en une désamination de cytidines à des sites spécifiques de l’ARNm. Trente-quatre sites d’édition ont été découverts dans les transcrits chloroplastiques d’Arabidopsis thaliana et plus de 500 dans les transcrits mitochondriaux. Depuis 2005, beaucoup de facteurs d’édition ont été trouvés. La majorité de ces protéines appartiennent à la famille des «PentatricoPeptide Repeat» (PPR). Parmi ces PPR, certaines contiennent un domaine DYW possédant de faibles similarités avec les cytidines désaminases (CDA), alors que d’autres en sont dénuées, générant un doute sur le fait qu’il ait une activité CDA. Le gène At1g47580 (DYW1) code une protéine unique chez Arabidopsis thaliana contenant «seulement» un domaine DYW. Il a été proposé que DYW1 puisse interagir avec les PPR ne contenant pas de domaine DYW, pour former un hétérodimère, capable d’éditer spécifiquement un site. En accord avec cette hypothèse, nous avons montré que DYW1 agissait sur le même site d’édition que CRR4, une PPR sans domaine DYW, et que ces protéines interagissaient in vivo. De plus, nous avons montré que DYW1 remplaçait les parties manquantes de CRR4 pour l’édition. Pour obtenir plus d’informations sur la fonction du domaine DYW, des mutations ont été introduites dans DYW1. Nous avons montré que la signature CDA dans les protéines DYW était essentielle à l’édition de l’ARN ainsi qu’à l’interaction avec les ions zinc. Les données sont en accord avec l’hypothèse d’une activité CDA dans le domaine DYW. Cependant, aucune activité CDA n’a pu être mise à jour in vitro. Il est vraisemblable qu’au moins un cofacteur doive encore être identifié. / In plant organelles, RNA editing mostly takes the form of conversions of cytidines to uridines at specific sites in mRNAs. Thirty-four editing sites have been found in Arabidopsis thaliana chloroplast transcripts and more than 500 sites in mitochondrial transcripts. Since 2005, lots of proteins have been found to act as RNA editing factors. Most of these proteins belong to the PentatricoPeptide Repeat (PPR) family. Amongst these PPR, some contain a DYW domain with weak similarity to cytidine deaminases (CDA), whilst others lack such a domain, creating doubts about whether this domain is required for editing. The gene At1g47580 (named DYW1) encodes a protein in Arabidopsis thaliana that contains “only” a DYW domain. Our initial hypothesis was that DYW1 might interact with PPR proteins that lack a DYW domain, in order to form a heterodimer, able to perform site-specific editing. In accordance with this hypothesis, we discovered that DYW1 is involved in editing the same site as CRR4, a PPR lacking a DYW domain, and that these two proteins interact together in vivo. Moreover, we showed that DYW1 replaces all the missing parts of CRR4 for editing. So, other partners need to be hypothesized for other DYW-lacking editing factors if this hypothesis is to be generalized. The highly conserved residues making up the CDA signature in DYW proteins were found to be essential for RNA editing and are also required for zinc binding, which is a known characteristic of CDAs. All the data so far are consistent with the DYW domain being (part of) a CDA activity; nevertheless, no CDA activity could be detected in vitro. It is likely that at least one required cofactor remains to be identified.
59

Molecular mechanisms of redoxin-mediated signalling in plant immunity

Kneeshaw, Sophie January 2016 (has links)
Posttranslational modification (PTM) of proteins is essential to creating a diverse proteome with the complex functions necessary to regulate key cellular processes. Redox-based PTMs exhibit many desirable characteristics to finely modulate transcriptional regulators; they occur rapidly and can alter protein conformation, localisation and activity. The plant immune system offers an excellent model in which to study redox-based modifications due to the rapid accumulation of oxidising agents that occurs during immune invasion. This so-called “oxidative burst” causes spontaneous oxidation of cysteine residues that are present in many regulatory proteins. These modifications fine-tune the activities of proteins that harbour them, enabling them to act in a concerted effort to reprogram the transcriptome, prioritising the expression of immune-related genes over housekeeping genes. Disulphide bonds (S-S) and S-nitrosothiols (SNO, i.e. the addition of an NO group to a cysteine moiety) have been shown to play particularly important roles in plant immunity. However, what still remains unclear is how these redox-based PTMs are rendered reversible, enabling them to act as molecular signalling switches. The work presented in this thesis explores a class of enzymes that are responsible for controlling the cellular levels of protein oxidation: the Thioredoxins. In addition to their well-established role in reducing disulphide bonds, I demonstrate in Chapter 3 that Thioredoxins are able to reverse protein S-nitrosylation during plant immune signalling. Immune-inducible Thioredoxin-h5 (TRXh5) was shown to be unable to restore immunity in gsnor1 mutants that display excessive accumulation of the NO donor S-nitrosoglutathione, but rescued impaired immunity and defence gene expression in nox1-mutants that exhibit elevated levels of free NO. This data indicates that TRXh5 discriminates between protein-SNO substrates to provide previously unrecognized specificity and reversibility to protein-SNO signalling in plant immunity. Furthermore, data is presented to show that TRXh5 reversed the effects of S.nitrosylation on many immune-related transcriptional regulators in vitro, forming the initial stages of an investigation into which proteins and pathways might be controlled by reversible S-nitrosylation in plant immunity (Chapters 3 & 4). Although the majority of transcriptional regulators are likely modified at their site of action, the nucleus, very little is currently known about nuclear redox signalling in plants. Therefore, in Chapter 5 a subclass of theThioredoxin superfamily was studied, the Nucleoredoxins, which have previously been shown to display disulphide reduction activity and localise in part to the nucleus. Here it is revealed that the activity and nuclear accumulation of Nucleoredoxin 1 (NRX1) is induced by the plant leaf pathogen Pseudomonas syringae, suggesting a key role for this protein in immune signalling. Target-capture experiments and subsequent mass spectrometry analysis identified the first in vitro targets of NRX1 and revealed many proteins with roles in oxidative stress, including the hydrogen peroxide scavenger Catalase 2 (CAT2). Moreover, overexpression of NRX1 was shown to be able to rescue the enhanced cell death phenotype of cat2 knockout mutants in response to the oxidative stressor, methyl viologen. Accordingly, nrx1 knockout mutants also exhibited an enhanced cell death phenotype in response to methyl viologen treatment. Together, these data indicate that NRX1 plays a key role in the control of oxidative stress-mediated cell death, potentially through direct regulation of Catalase proteins. Taken together, the work in this thesis implicates members of the Thioredoxin family as key regulators of transcriptional reprogramming during plant immunity and uncovers a novel role for Thioredoxin superfamily member, NRX1, in the control of oxidative stress.
60

Distinct transcriptional signatures of aneuploidy in murine pluripotent cell populations

Skylaki, Stavroula January 2012 (has links)
Genomic integrity in mouse embryonic and induced pluripotent stem cells can be compromised by factors such as extended time in culture and cellular reprogramming. Surprising, only a few studies have thus far examined the accumulation of chromosomal imbalances in mouse pluripotent populations upon prolonged propagation in vitro. It is presumed that specific recurring genetic changes can confer selective growth advantage and resistance to apoptosis and/or differentiation to the affected cells, although the genes that drive these processes remain elusive. The presence of these changes in published studies can confound the analysis of the data and hinder the reproducibility of the results. At the transcriptional level, aneuploidy manifests as large chromosomal regions of aberrant gene expression. This thesis presents a method to identify these regions in large-scale datasets and interrogate for recurrent patterns. The present analysis shows that over half of the 315 mouse pluripotent samples examined carry whole or partial-chromosome spanning clusters of aberrant transcription. Furthermore, there are common gene expression changes across samples with any type of predicted aneuploidy and samples with chromosome-specific aberrations. These transcriptional signatures have been used to train classification models which can predict aneuploid samples with over 90% accuracy. This is an important step towards the development of a low-cost and reliable transcriptional validation assay for the presence of aneuploidy.

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