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Kou Ying Chuck. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 186-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Establishment of the biochemical and molecular nature of cardiac development is essential for us to understand the relationship between genetic and morphological aspects of heart formation. The molecular mechanisms that underly heart development are still not clearly defined. To address this issue we have used two approaches to identify genes involved in early chick cardiac development. Differential display previously conducted in our laboratory led to the identification of two gene fragments differentially expressed in the heart that are further described in this thesis. The full-length cDNA sequence of both eukaryotic translation initiation factor-2b (eIF-2b) and NADH cytochrome b5 reductase (b5R) were isolated using library screening. The upreglation of these genes during heart development is expected given the heart is the first functional organ to form in vertebrates and protein synthesis and cell metabolism at this stage of development is maximal. Limitations in the differential display approach led to the development and optimisation of a subtractive hybridisation approach for use with small amounts of cells or tissue. To focus on cardiac gene expression during the initial phases of heart development, subtractive hybridization was performed between the cardiogenic lateral plate mesoderm of Hamburger and Hamilton stage 4 embryos and the heart primordia of stage 9 embryos. Of the 87 independent clones identified by this procedure, 59 matched known sequences with high homology, 25 matched unknown expressed sequence tag (EST) sequences with high homology, and 3 did not match any known sequence on the database. Known genes isolated included those involved in transcription, translation, cell signalling, RNA processing, and energy production. Two of these genes, high mobility group phosphoprotein A2 (HMGA2) and C1-20C, an unknown gene, were chosen for further characterisation. The role of each gene in early chick heart development and indeed development in general, was addressed using techniques such as in situ hybridisation, transfection analysis, in ovo electroporation and RNAi. HMGA2 is a nuclear phosphoprotein commonly referred to as an architectural transcription factor due to its ability to modulate DNA conformation. In keeping with this function, HMGA2/GFP fusion protein was shown to localise to the nucleus and in particular, the nucleolus. In situ hybridisation analysis suggested a role for HMGA2 in heart and somite development. HMGA2 expression was first detected at HH stage 5 in the lateral plate mesoderm, a region synonymous with cells specified to the cardiac fate. HMGA2 was also strongly expressed in the presomitic segmental plate mesoderm and as somites developed from the segmental plate mesoderm, the expression of HMGA2 showed an increasingly more restricted domain corresponding to the level of maturation of the somite. Restriction of HMGA2 expression was first detected in the dorsal region of the epithelial somite, then the dorsomedial lip of the dermomyotome, and finally the migrating epaxial myotome cells. The novel intronless gene, C1-20C, predicts a protein of 148 amino acids containing a putative zinc finger binding domain and prenyl binding motif. Zinc binding assays showed that the zinc finger domain of C1-20C/MBP fusion protein bound over six times the quantity of zinc compared to MBP alone, although not in a 1:1 stoichiometric molar ratio. C1-20C/GFP fusion protein was shown to localise to as yet unidentified intracellular cytoplasmic vesicular compartments. These compartments did not colocalise with the endosome/lysosome pathway, aparently ruling out a role for C1-20C in protein trafficking, recycling or degradation. Expression of C1-20C in the chick embryo suggests a possible role in heart and notochord development and preliminary results using siRNA suggest that C1-20C is involved in normal heart looping.

Chez les eucaryotes pluricellulaires, la différenciation des cellules repose en partie sur l'activation oula répression des gènes. Les profils d'expression génique mis en place vont perdurer d'une générationcellulaire à l'autre. Ce phénomène met en jeu des mécanismes épigénétiques qui remodèlentlocalement la structure de la chromatine. Chez Drosophila melanogaster, les protéines des groupesPolycomb (PcG) et Trithorax (TrxG) participent au maintien du profil d'expression des gènes au coursdu développement. Les protéines PcG maintiennent les gènes réprimés tandis que les protéines TrxGmaintiennent les gènes activés. Une troisième classe de protéines nommée Enhancers of Trithoraxand Polycomb (ETP) module l'activité des PcG et TrxG. Dorsal Switch Protein 1 (DSP1) est uneprotéine HMGB (High Mobility Group B) classée comme une ETP. Par tamisage moléculaire, nousavions montré que la protéine DSP1 était présente au sein de complexes de poids moléculaire de 100kDa à 1 MDa. Le travail de thèse présenté ici a pour but d'identifier les partenaires de la protéineDSP1 dans l'embryon et de mieux connaître les propriétés biochimiques de DSP1. Premièrement, j'aimis en place puis effectué l'immunopurification des complexes contenant DSP1 dans des extraitsprotéiques embryonnaires. Cette approche nous a permis d'identifier 23 partenaires putatifs de laprotéine DSP1. Parmi ces protéines, nous avons identifié la protéine Rm62 qui est une ARN hélicaseà boîte DEAD. Les relations biologiques entre DSP1 et Rm62 ont été précisées. Deuxièmement, j'aidéterminé, par une approche biochimique, de nouvelles caractéristiques physico-chimiques de laprotéine DSP1.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (p. 68-76).

Orientador: Celso Eduardo Benedetti / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:56:52Z (GMT). No. of bitstreams: 1 Souza_TiagoAntoniode_M.pdf: 8040684 bytes, checksum: 0b9836df8d96343f09503df5056436cd (MD5) Previous issue date: 2010 / Resumo: O cancro cítrico, causado pela bactéria Xanthomonas axonopodis pv. citri (Xac), é uma doença que afeta a maioria das espécies do gênero Citrus, ocorrendo praticamente em todos os continentes, e se destaca como uma das ameaças à citricultura brasileira. O mecanismo molecular pelo qual Xac causa o cancro não é inteiramente conhecido, entretanto, sabe-se que a bactéria ao infectar a planta, utiliza o sistema secretório tipo ??? (TTSS) para injetar proteínas de patogenicidade, entre elas PthAs da família AvrBs3/PthA. Quando expresso na célula hospedeira, PthA induz lesões características do cancro como hipertrofia e hiperplasia. Estudos recentes demonstram que membros dessa família atuam como fatores de transcrição. Portanto, a elucidação de como PthA ativa a transcrição é de grande importância para o entendimento do seu mecanismo de ação e desenvolvimento das lesões do cancro. Neste contexto, o presente projeto teve como objetivo caracterizar interações entre a proteína PthA de Xac e as proteínas CsARF (Auxin Response Factor) e CsHMG (High-mobility group) de laranja doce (Citrus sinensis), previamente identificadas em ensaios de duplo híbrido de leveduras. CsARF tem elevada similaridade com AtARF2, um repressor transcricional envolvido na via de sinalização por auxinas. Hormônios vegetais desempenham um importante papel na interação planta-patógeno e em nosso laboratório verificamos que auxinas são importantes para o desenvolvimento dos sintomas do cancro. CsARF foi capaz de interagir com a maioria das variantes de PthA tanto in vitro quanto em ensaios de duplo-híbrido de leveduras. A interação de CsARF com PthA se dá através dos domínios C-terminal Aux/IAA e B3 de ligação ao DNA. Verificamos que o promotor do gene de uma expansina de citros, induzido por Xac e auxina, apresenta possíveis sítios de ligação das proteínas CsARF e PthA. Dados de EMSA indicam que PthA e CsARF ligam em sítios adjacentes no promotor da expansina de citros e que a interação de PthA com CsARF poderia deslocá-la do promotor. A proteína CsHMG é semelhante a AtHMGB1 de Arabidopsis thaliana, envolvida em crescimento celular. CsHMG interagiu com todas as variantes de PthA, sendo que essa interação envolve uma região rica em leucinas (LRR), idêntica nas quatro variantes de PthA. Verificou-se também que CsHMG é capaz de ligar DNA de forma inespecífica. Por outro lado, CsHMG ligou RNA in vitro, com especificidade para RNAs ricos em uridina (poly-U). Como PthA age como fator de transcrição eucarioto, não é surpreendente que proteínas do hospedeiro envolvidas com regulação gênica sejam capazes de interagir com esse efetor, sugerindo um novo modo de ação de proteínas efetoras bacterianas. / Abstract: Citrus canker disease, caused by Xanthomonas axonopodis pv. citri (Xac), affects almost all citrus species and represents a major threat to the Brazilian citriculture. The molecular mechanism by which Xac causes citrus canker disease is poorly understood, however the bacterium injects pathogenicity proteins through a type III secretion system (TTSS) including proteins of AvrBs3/PthA family proteins. When transiently expressed in host cells, PthAs alter transcription of the host cell to the benefit of the pathogen, leading to the development of the cancer lesions, including hypertrophy and hyperplasia. These proteins are thought to acts as eukaryotic transcriptional factors, binding and activating directly promoters of host genes. Therefore, elucidating how activates PthA transcription is very important to understanding the mechanisms governing the development of canker lesions. To elucidate how PthA activates transcription and to establish its molecular mode of action, a two-hybrid approach was used to identify host proteins that interact with PthA and therefore could be important for the development of the canker lesions. Among the citrus proteins identified, we selected for studies a CsARF (Auxin Response Factor) and a CsHMG (High-mobility group), both involved in regulation of gene transcription. CsARF shares high similarity to the Arabidopsis thaliana ARF2, involved in the auxin signaling pathway. This is in line with our previous studies showing that auxin is required for canker development. The interactions between all variants of PthA were analyzed both in vivoand in vitro and depend on the repeat domain of PthAs. The B3 DNA binding and the Aux/IAA domains of CsARF are both involved in protein-protein interactions. Interestingly, the citrus promoter of a citrus expansin gene that is up-regulated by Xac and auxin contains putative CsARF and PthA binding sites. Since these sites are located adjacent in this promoter, it is suggested that the interaction of PthA with CsARF might somehow affect the regulation of the expansin promoter. CsHMG is highly similar to the A. thaliana HMGB1 involved in cell growth. CsHMG interacts with all PthA variants and its interaction was shown to be mediated primarly by the leucine-rich repeat (LRR) region of PthAs. CsHMG binds to DNA in a non-specific fashion; surprisingly, however, CsHMG shows an as yet unreported ability to bind to synthetic RNA forms with an apparent specificity to poly-U probes. PthA acts like an eukaryotic transcription factor and is not surprising that host proteins involved with gene regulation can interact with this effector, suggesting a new mode of action of these bacterial effector proteins. / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

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Actuellement le polyéthylènimine (PEI) est l’agent de transfection transitoire le plus utilisé par l’industrie pharmaceutique pour la production de protéines recombinantes à grande échelle par les cellules de mammifères. Il permet la condensation de l’ADN plasmidique (ADNp) en formant spontanément des nanoparticules positives appelées polyplexes, lui procurant la possibilité de s’attacher sur la membrane cellulaire afin d’être internalisé, ainsi qu’une protection face aux nucléases intracellulaires. Cependant, alors que les polyplexes s’attachent sur la quasi-totalité des cellules seulement 5 à 10 % de l’ADNp internalisé atteint leur noyau, ce qui indique que la majorité des polyplexes ne participent pas à l’expression du transgène. Ceci contraste avec l’efficacité des vecteurs viraux où une seule particule virale par cellule peut être suffisante. Les virus ont évolués afin d’exploiter les voies d’internalisation et de routage cellulaire pour exprimer efficacement leur matériel génétique. Nous avons donc supposé que l’exploitation des voies d’internalisation et de routage cellulaire d’un récepteur pourrait, de façon similaire à plusieurs virus, permettre d’optimiser le processus de transfection en réduisant les quantités d’ADNp et d’agent de transfection nécessaires. Une alternative au PEI pour transfecter les cellules de mammifèreest l’utilisation de protéines possédant un domaine de liaison à l’ADNp. Toutefois, leur utilisation reste marginale à cause de la grande quantité requise pour atteindre l’expression du transgène. Dans cette étude, nous avons utilisé le système E/Kcoil afin de cibler un récepteur membranaire dans le but de délivrer l’ADNp dans des cellules de mammifères. Le Ecoil et le Kcoil sont des heptapeptides répétés qui peuvent interagir ensemble avec une grande affinité et spécificité afin de former des structures coiled-coil. Nous avons fusionné le Ecoil avec des protéines capables d’interagir avec l’ADNp et le Kcoil avec un récepteur membranaire que nous avons surexprimé dans les cellules HEK293 de manière stable. Nous avons découvert que la réduction de la sulfatation de la surface cellulaire permettait l’attachement ciblé sur les cellules par l’intermédiaire du système E/Kcoil. Nous démontrons dans cette étude comment utiliser le système E/Kcoil et une protéine interagissant avec l’ADNp pour délivrer un transgène de manière ciblée. Cette nouvelle méthode de transfection permet de réduire les quantités de protéines nécessaires pour l’expression du transgène. / Pharmaceutical industry often employs polyethylenimine (PEI) for large scale protein production processes by transient transfection of mammalian cells. PEI condenses plasmid DNA (pDNA) by spontaneously forming positive nanoparticles known as polyplexes. Condensed pDNA is favoured for cell surface binding, internalization and protection from intracellular nucleases. While most of the cells efficiently uptake polyplexes, only 5 to 10% of captured pDNA reaches the nucleus for transgene expression. This suggests that polyplexes are hampered in their ability to route and to translocate to the nucleus necessitating large amounts of polyplexes to achieve high expression levels. By contrast, many viruses can efficiently transduce cells with only one or a few viral genome copies. Viruses have evolved to exploit cellular internalization and routing properties to express their own genetic material. We hypothesized that less pDNA would be used in an optimized transfection process if we exploited the internalization and routing properties that viruses use. DNA binding proteins could be used as an alternative to PEI to transfect mammalian cells. However, their usage is marginal due to the large protein quantities required to bind pDNA for transgene expression. If less pDNA is used less binding protein is needed. In this study, we used the E/Kcoil system to target a membrane receptor to deliver pDNA in mammalian cells. The Ecoil and Kcoil are two repeated heptapeptides which interact with a high affinity and specificity to form coiled-coil structures. We fused the Ecoil with a recombinant pDNA-binding protein. The Kcoil was fused to a stably-expressed membrane receptor in HEK293 cells. We discovered that low sulfation of the cell surface reduced non-specific binding of the pDNA:protein complex and permitted targeted binding via the E/Kcoil interaction. We demonstrate how to use recombinant pDNA-binding protein and the E/Kcoil system for targeted transgene delivery. This newly developed system provides a new transfection method, with reduced pDNA-binding protein quantities needed to achieve transgene expression.

La poly(ADP-ribosyl)ation est une modification post-traductionnelle des protéines catalysée par une famille d’enzymes : les poly(ADP-ribose) polymérases. Parmi les plus étudiées, PARP-1 et PARP-2 interviennent dans l’organisation, l’expression et le maintien de l’intégrité du génome. Nous avons initié l'étude d'un nouveau partenaire de PARP-1 préalablement identifié par double-hybride, et encore peu étudié à ce jour : HMG2L1 (High-Mobility Group protein 2 Like-1). La protéine humaine de 601 acides aminés contient un domaine HMG (High-Mobility Group) normalement impliqué dans l’interaction avec l’ADN. Quelques études ont montré que HMG2L1 régule la transcription en agissant comme co-régulateur négatif ou positif. Dans un premier temps, nous avons caractérisé le lien entre PARP-1 et HMG2L1. L’interaction avec PARP-1 a été confirmée in-vivo et in vitro. Nous avons montré que HMG2L1 pouvait également interagir avec PARP-2. HMG2L1 est poly(ADP-ribosyl)ée par PARP-1 et PARP-2, de même qu’elle est capable d’interagir avec le poly(ADP-ribose). La construction de formes tronquées de HMG2L1 en fusion avec la GFP nous a permis de montrer que le domaine N-terminal – en amont du domaine HMG – est impliqué dans ces interactions. Ce domaine N-terminal est très électropositif et intrinsèquement désordonné ce qui lui confère de nombreuses potentialités d’interactions. L’expression des fusions GFP dans des cellules HeLa nous a permis de montrer la localisation nucléaire et nucléolaire de HMG2L1, comme c’est le cas pour PARP-1 et PARP- 2. En outre, HMG2L1 colocalise avec UBF (Upstream Binding Factor), le facteur de transcription de l’ARN polymérase I responsable de la transcription des ARN ribosomaux. La surexpression de GFP-hHMG2L1 entraîne un stress nucléolaire caractérisé par l’inhibition de la transcription des ADNr et la formation de coiffes nucléolaires. Nous avons également entrepris une recherche de partenaires de HMG2L1 par spectrométrie de masse. De nombreuses protéines nucléolaires, impliquées dans la biogenèse des ribosomes ou la maturation des ARNs ont été identifiées, suggérant un rôle de HMG2L1 dans ces processus. Nous avons montré que la protéine purifiée interagit avec l’ADN via son domaine HMG principalement, et qu’elle interagit avec l’ARN via son domaine N-terminal. Mais surtout, nous avons mis en évidence une activité ARN-chaperonne, qui peut être régulée par le poly(ADP-ribose). La localisation de HMG2L1, son réseau d’interaction ainsi que son activité chaperonne nous laissent à penser qu’elle pourrait être impliquée dans des processus de maturation des ARN, régulés par la poly(ADPribosyl)ation. / Poly(ADP-ribosyl)ation is a post-translational modification of proteins mediated by a family of enzymes called poly(ADP-ribose) polymerases. Among the best studied, PARP-1 and PARP-2 are both implicated into the transcription, organization and integrity of genome. We have initiated the characterization of a new PARP-1 partner previously identified in a yeast two-hybrid screen, and still poorly studied: HMG2L1 (High-Mobility Group protein 2 Like-1). The human protein of 601 amino acids contains one HMGbox domain normally implicated in the recognition of DNA. Some studies have reported the role of HMG2L1 in the regulation of transcription by acting as a negative or positive coregulator. First, we characterized the link between PARP-1 and HMG2L1. We confirmed the interaction between both proteins in vivo and in vitro. We also showed that HMG2L1 couldinteract with PARP-2. HMG2L1 is poly(ADP-ribosyl)ated by PARP-1 and PARP-2, and is able to interact with poly(ADP-ribose). The construction of GFP-fused truncated versions of HMG2L1 allowed us to show that the N-terminal part – upstream to the HMGbox – is responsible for all these interactions. This N-terminal domain is highly electropositive and intrinsically disordered conferring a lot of interactions potentialities. The expression of the GFP-fused proteins in HeLa cells allowed us to localizeHMG2L1 into the nucleus and the nucleolus, like PARP-1 and PARP-2. Moreover, HMG2L1 colocalizes with UBF (Upstream Binding Factor), the transcription factor responsible for the transcription of ribosomal ARNs by RNA polymerase I. The overexpression of GFPhHMG2L1 leads to a nucleolar stress illustrated by the inhibition of transcription and the formation of nucleolar caps. We also undertook a proteomic study to find new partners of HMG2L1. We found a huge amount of nucleolar proteins, involved in ribosome biogenesis or RNA maturation, suggesting that HMG2L1 could be involved in these processes. Finally, we demonstrated the ability of the purified protein to interact with DNA mostly through its HMGbox domain and RNA through its N-terminal domain. Moreover, we discovered that HMG2L1 is endowed with a RNA-chaperone activity, that can be regulated by poly(ADP-ribose). Taken together, the localization of HMG2L1, its interacting partners and its RNA chaperone activity allow us to make the assumption that HMG2L1 could be implicated in RNA maturation processes, regulated by poly(ADP-ribosyl)ation.