Orientador: Michel Georges Albert Vincentz / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-09T01:42:08Z (GMT). No. of bitstreams: 1 Silveira_AmandaBortolini_M.pdf: 20873886 bytes, checksum: 0c34c8a141f7bb11f8f3176ba7a976e0 (MD5) Previous issue date: 2007 / Resumo: O crescimento e o desenvolvimento dos organismos são baseados na capacidade celular de expressão gênica diferencial que resulta, principalmente, do controle da taxa de iniciação da transcrição por fatores reguladores de transcrição (FTs). FTs do tipo Basic Leucine QQjJer(bZIP) fQram descritos em todos os eucariotos. Seu domínio conservado é constituído de uma região de ligação ao DNA rica em aminoácidos básicos, flanqueada a um zíper de leucinas responsável pela dimerização. Em angiospermas, os bZIPs são reguladores importantes de processos específicos como fotomorfogênese, desenvolvimento de órgãos, elongação celular, controle do balanço de carbono/nitrogênio, mecanismos de defesa, via de sinalização de hormônios e sacarose, controle osmótico e florescimento. Mostramos que os genomas de Arabidopsis thaliana e Orysa sativa codificam para um conjunto completo e não redundante de 76 e 113 fatores bZIP respectivamente, que foram organizados em 11 grupos de proteínas evolutivamente relacionadas e 33 Possíveis Grupos de Genes Ortólogos (PoGO) de mono e eudicotiledôneas, o que deve permitir racionalizar o processo de caracterização funcional destes fatores em angiospermas. O Grupo C, que inclui genes homólogos ao lócus de regulação Opaco-2 (02) de milho, está organizado em três PoGOS, que possivelmente desempenham três funções ancestrais de angiospermas. Em Arabidopsis estas três possíveis funções ancestrais estão representadas por quatro genes (bZIP' 02 !1omologous, Bzo2h), Bzo2h3/AtbZIP63 (PoGO C1), Bzo2h1/AtbZIP10 e Bzo2h4/ AtbZIP25 (PoGO C2) e Bzo2h2/AtbZIP9 (PoGO C3). Visando um melhor conhecimento sobre a evolução das funções dos fatores bZIP de angiospermas do Grupo C, iniciamos a caracterização funcional de~tes quatro reguladores, focando principalmente em AtbZIP9, um gene único representativo de uma função ancestral de angiospermas e cujo papel ainda é desconhecido. Notamos que a expressão de AtbZIP9 é restrita as células do floema e regulada por glicose, ácido abscísico e citocinina, sugerindo que este gene integra as vias de sinalização destes sinais metabólicos e hormonais no floema. Abordagens de genética reversa como RNAi, knockout e superexpressão não permitiram elucidar de maneira clara a atuação de AtbZIP9 no ciclo de vida de Arabidopsis, indicando que mecanismos de regulação pós-transcricional e/ou redundância genética atuam sobre este gene. Visando dar continuidade e ampliar o estudo funcional de AtbZIP9, foram obtidas linhagens transgênicas de Arabidopsis expressando versões modificadas deste gene que codificam para proteínas ativadoras constitutivas fortes da transcrição. Estas novas versões de AtbZIP9 são teoricamente capazes de ativar de maneira constitutiva a expressão dos genes alvos de AtbZIP9, contornando assim, as dificuldades decorrentes da análise de famílias gênicas que apresentam redundância funcional. Quando comparados a plantas selvagens, transformantes primários para ativadores constitutivos fortes apresentaram diversas alterações de morfologia foliar, além de mudanças metabólicas e fisiológicas como acúmulo de compostos fenólicos em folhas, sintomas de morte celular e senescência. A análise destes transformantes ainda sugere uma possível participação de AtbZIP9 no controle do desenvolvimento do sistema vascular de raízes e folhas. Suspeitamos que as alterações de morfologia foliar e fisiologia observadas possivelmente representem conseqüências de mudanças nas propriedades funcionais de transporte do floema, decorrentes de defeitos no processo de diferenciação e organização das células do cilindro vascular / Abstract: Transcriptional regulatory factors (TFs) play an important role in controlling growth and development of ali organisms. bZIPs TFs have been described in ali eukaryotes and are characterized by a basic aminoacid rich DNA binding domain and a leucine zipper, responsible for dimerization. bZIPs have been reported to act in several different plantspecific processes such as organ development, cell elongation, defense mechanism, hormones and sucrose signalization, light response, control of nitrogen/carbon balance, osmotic control and flowering. We showed that Arabidopsis thaliana and Orysa sativa genomes encode a complete and non-redundant set of 76 and 113 bZIP transcription factors, respectively, which were divided into 11 unique groups of homologous genes. More detailed phylogenetic analysis led to the identification of 33 Possible Groups of Monocot and Eudicot Orthologous Genes (PoGO), which allows rationalizing functional studies in angiosperms. Group C, which includes genes homologous to the maize Opaque-2 locus, is formed by three PoGOs, suggesting that this group represents three ancestral functions among angiosperms. In Arabidopsis these three possible ancestral functions may be represented by the bZIP Qpaque-2. homologous genes (Bzo2h), Bzo2h3/AtbZIP63 (PoGO C1), Bzo2h1/AtbZIP10 and Bzo2h4/AtbZIP25 (PoGO C2) and Bzo2h2/AtbZIP9 (PoGO C3). To get insight into the evolution pattern and function of Group C members, we have iniciated the functional characterization of the Bzo2h genes concentrating initially on AtbZIP9, a unique gene that represents an ancestral function and for which no functional informational is available. We showed that AtbZIP9 expression is restricted to phloem cells and regulated by glucose, abscisic acid and cytokinin, suggesting that this gene is an element of the signalization pathways of these metabolic and hormonal signals in the phloem. Reverse genetic approaches such as RNAi, knockout and superexpression failed to reveal the biological function of AtbZIP9 in Arabidopsis life cycle and suggested that post-transcriptional regulation and/or functional redundancy may act on AtbZIP9. In order to improve our Rnowledge on AtbZIP9 function, Arabidopsis transgenic lines expressing constitutive transcriptional activator versions of AtbZIP9 were obtained. Since such modified versions of AtbZIP9 are theoretically able to promote the superexpression of AtbZIP9 target genes, this strategy should be independent of functional redundancy. When compared to wild type plants, primary transformants for constitutive transcriptional activator versions of AtbZIP9 showed alterations of leaf morphology, as well as metabolic and physiologic modifications, such as phenolic compound accumulation in leaves, cell death and senescence symptoms. Analyses of this transformants also suggest that AtbZIP9 is possibly involved in the control of leaf and root vascular system development. We suspect that the alteration of leaf morphology and physiology observed in primary transformants possibly reflects consequences of changes in phloem transport functional properties, due to defects in vascular cylinder cell differentiation and organization / Mestrado / Genetica Vegetal e Melhoramento / Mestre em Genética e Biologia Molecular

Orientadores: Ricardo Aparicio, Marcelo Menossi Teixeira / Dissertação (mestrado) - Universidade Esstadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-12T12:36:03Z (GMT). No. of bitstreams: 1 Kiyota_Eduardo_M.pdf: 3864663 bytes, checksum: fb929727fccb0492142f162a1eca404e (MD5) Previous issue date: 2008 / Resumo: Os fatores de transcrição do tipo bZIP estão presentes em organismos eucariotos e estão envolvidos na regulação da expressão gênica e no controle de muitos processos intracelulares. Esses fatores se ligam a seqüências específicas no DNA e são capazes de reconhecer seqüências reguladoras no promotor de um gene. As bZIPs são caracterizadas por uma região conservada rica em resíduos de aminoácidos básicos, e um zíper de leucinas, que possui repetições de uma seqüência de aminoácidos hidrofóbicos onde há uma leucina que ocupa a mesma posição a cada 7 resíduos. Estudos estruturais com bZIPs mostraram que essas proteinas enovelam-se na forma de uma extensa hélice-a e são capazes de formar dímeros através de um arranjo do tipo coiled- coil. Neste trabalho, a parte correspondente à região básica e ao zíper de leucinas de duas bZIPs, SCF5 e SCF12 de cana-de-açúcar, pertencentes a sub-famílias diferentes, foram clonadas, expressas e purificadas para estudos estruturais. O DNA correspondente a SCF12 foi clonado em pET28a e a proteína recombinante foi produzida em E. coli BL21 (DE3) pRil. A SCF12 purificada por cromatografia de afinidade (IMAC) teve sua estrutura secundária caracterizada por dicroísmo circular. A SCF5, clonada em pET3C e expressa em E. coli BL21 (DE3) pLysS foi purificada por cromatografia de troca catiônica. Cristais de um complexo da proteína ligada a uma seqüência de DNA de 24 pares de bases foram obtidos mas não exibiram qualidade suficiente para permitir a determinação da estrutura cristalográfica. Entretanto, foi possível obter um modelo do complexo a partir de experimentos de espalhamento de Raios X a baixos ângulos (SAXS, do inglês Small Angle X-Ray Scattering) em solução, e interpreta-lo à luz de estruturas de homólogas já conhecidas. / Abstract: The bZIP transcription factors are present in eukaryotic organisms and are involved in the regulation of gene expression and many intracellular processes. These factors bind specific DNA sequences and are able to recognize regulatory sequences of a gene promoter. The bZIPs are characterized by a conserved region rich in basic amino acid residues as well as by having the leucine zipper region, which possess a sequence of hydrophobic residues where there are leucines every seventh amino acids. Structural studies have shown that bZIP-folding is alpha-helical and these proteins are capable of dimmer formation via coiled-coil arrangement. In this work, the basic region and the leucine zipper of two sugarcane bZIPs, SCF12 and SCF5, belonged to two different bZIP-families were cloned, expressed and purified for structural studies. The corresponding SCF12 DNA was cloned into pET28a expression vector and the protein was produced in E. coli BL21 (DE3) pRil cells. SCF12 protein was purified by affinity chromatography (IMAC) and had its secondary structure characterized by CD. SCF5, cloned into pET3c and expressed in E. coli BL21 (DE3) pLysS was purified by cation exchange chromatography. Crystals of a complex formed by SCF5 protein and a 24-base-pair DNA sequence were obtained but unfortunately with quality insufficient for crystallographic structure determination. However, it was possible to obtain a model of the analyzed complex applying Small Angle X-ray Scattering (SAXS) technique by protein homologous structure comparison. / Mestrado / Físico-Química / Mestre em Química

Les protéines GILZ (Glucocorticoid-Induced Leucine Zipper) et TSC-22 (Transforming growth factor-beta Stimulated Clone-22) appartiennent à la famille de protéines TSC-22D (TSC-22 Domain). GILZ a été décrit précédemment comme étant induit au cours de la déprivation en interleukine-2 (IL-2) des lymphocytes de la lignée cellulaire CTLL-2, permettant ainsi de retarder leur apoptose. Le but de notre travail était de déterminer les rôles respectifs de GILZ et TSC-22 au cours de l’apoptose des cellules CTLL-2.Nos résultats ont permis de montrer que TSC-22 augmentait l’apoptose induite par la déprivation en IL-2 des cellules CTLL-2. Nous avons mis en évidence une augmentation de l’activation des caspases ainsi qu’une régulation positive de l’expression de BIM. Nous avons en outre montré que l’expression de GILZ, protéine anti-apoptotique, induite lors de la déprivation en IL-2, était régulée négativement en présence de TSC-22. Enfin, nous avons montré que l’expression de l’ARNm de gilz était régulée négativement par TSC-22, mais que la stabilité de son ARNm n’était pas modifiée.Notre travail a donc permis de montrer que TSC-22 accélère l’entrée en apoptose des lymphocytes T en régulant négativement l’expression de la protéine anti-apoptotique GILZ. / GILZ (Glucocorticoid-Induced Leucine Zipper) and TSC-22 (Transforming growth factor-beta Stimulated Clone-22) belong to the TSC-22D (TSC-22 Domain) family of proteins. GILZ has been previously shown to be induced upon interleukin-2 (IL-2) deprivation in the T-cell line CTLL-2, allowing cells to delay apoptosis. The aim of our study was to elucidate the respective roles of GILZ and TSC-22 during IL-2 deprivation-induced T-lymphocytes apoptosis.Our results demonstrated that TSC-22 increased CTLL-2 cells apoptosis induced upon IL-2 deprivation. We highlighted in TSC-22 expressing cells both an increase in caspases activation and BIM expression up-regulation. We also demonstrated that GILZ expression, an anti-apoptotic protein, known to be induced after IL-2 withdrawal, was down-regulated in the presence of TSC-22. Moreover, we showed that gilz mRNA expression was also significantly repressed, but gilz mRNA half-life was not modified.Altogether, these results suggest that, in T-cells, TSC-22 could behave as a repressor of GILZ expression, accelerating IL-2 deprivation-induced apoptosis.

Une cellule dendritique (CD) qui exprime le facteur de transcription GILZ durant l'apprêtement de l'antigène et sa présentation aux cellules effectrices, génère des lymphocytes T régulateurs (LTregs) CD25high Foxp3+ sécréteurs d'IL-10. La production de GILZ est dépendante de l'action des glucocorticoïdes, de l'IL-10 et du TGF-.Nous avons mis en évidence chez l'homme qu'une corticothérapie orale de 48h induit l'expression de GILZ dans les cellules présentatrices de l'antigène circulantes (CPAs) de sujets allergiques. Les CPAs isolées après la corticothérapie génèrent des LTregs CD25high Foxp3+ IL-10+ spécifiques de l'allergène.Nous également constaté in vitro que les mastocytes participent à l'activation des CDs au cours des réactions allergiques en régulant l'expression de GILZ. Les médiateurs d'origine mastocytaire, dont l'histamine, diminuent l'expression de GILZ dans les CDs et altèrent ainsi leur capacité à activer des LTregs. Nous avons identifié le mécanisme par lequel l'histamine diminue l'expression de GILZ dans les CDs humaines. L'histamine inhibe l'activité transcriptionnelle de Foxo3, un facteur de transcription régulant l'expression de GILZ. Enfin, nous avons démontré que des souris transgéniques qui surexpriment GILZ constitutivement dans les CDs sont protégées contre le développement de l'asthme allergique. L'ensemble de ces résultats permet d'envisager de nouvelles stratégies d'immunomodulation dans l'allergie, centrée sur la régulation de l'expression de GILZ dans les CDs.

Les protéines GILZ (Glucocorticoid-Induced Leucine Zipper) et TSC-22 (Transforming growth factor-beta Stimulated Clone-22) appartiennent à la famille de protéines TSC-22D (TSC-22 Domain). GILZ a été décrit précédemment comme étant induit au cours de la déprivation en interleukine-2 (IL-2) des lymphocytes de la lignée cellulaire CTLL-2, permettant ainsi de retarder leur apoptose. Le but de notre travail était de déterminer les rôles respectifs de GILZ et TSC-22 au cours de l'apoptose des cellules CTLL-2.Nos résultats ont permis de montrer que TSC-22 augmentait l'apoptose induite par la déprivation en IL-2 des cellules CTLL-2. Nous avons mis en évidence une augmentation de l'activation des caspases ainsi qu'une régulation positive de l'expression de BIM. Nous avons en outre montré que l'expression de GILZ, protéine anti-apoptotique, induite lors de la déprivation en IL-2, était régulée négativement en présence de TSC-22. Enfin, nous avons montré que l'expression de l'ARNm de gilz était régulée négativement par TSC-22, mais que la stabilité de son ARNm n'était pas modifiée.Notre travail a donc permis de montrer que TSC-22 accélère l'entrée en apoptose des lymphocytes T en régulant négativement l'expression de la protéine anti-apoptotique GILZ.

La leucine zipper de la rétine neurale (Nrl) joue un rôle central dans le développement et l'homéostasie des bâtonnets en activant I'expression de gènes tels que le photopigment Rhodopsine. Nrl est aussi associé à la Rétinite Pigmentaire, faisant ainsi de ce gène un modèle intéressant pour la compréhension des programmes contrôlant le développement et I'homéostasie des photorécepteurs.Ce travail de thèse vise à caractériser les mécanismes régulateurs de I'expression de Nr/ au cours du développement rétinien. L'électroporation in vivo de vecteurs rapporteurs dans des rétines de souris en développement, a révélé des séquences minimales de promoteur Nr/ nécessaires à une expression spécifique dans les photorécepteurs. Nous avons identifié RORI3 comme facteur requis pour cette expression, et montré que les facteurs OTX2, CRX et CREB s'accrochent aussi directement à des régions régulatrices particulières du promoteur. Nous avons construit un virus adéno-associé (AAV) contenant un promoteur minimal Nrl de 0.3 kb, et montré qu'il est adapté à la délivrance de gène spécifiquement dans les photorécepteurs.Nous avons montré que NRL, CRX et NR2E3, les régulateurs principaux de la Rhodopsine, ont une expression rythmique au cours de 24 h, et que l'expression cyclique de Nr/ peut être due à l'activation par RORp, un composant l'horloge circadienne. Enfin, nous avons identifié un nouveau facteur de transcription, NonO, au niveau de la région du promoteur proximal de la Rhodopsine, qui en combinaison avec NRL et CRX, active le promoteur de la Rhodopsine. L'invalidation de NonO au cours du développement rétinien a prouvé son implication pour le développement et I'homéostasie des bâtonnets.

DNA damage response pathways are a complicated network of proteins that function to remove and/or reverse DNA damage. Following genetic insult, a signal cascade is generated, which alerts the cell to the presence of damaged DNA. Once recognized, the damage is either removed or the damaged region is excised, and the original genetic sequence is restored. However, when these pathways are defective the cell is unable to effectively mediate the DNA damage response and the damage persists unrepaired. Thus, the proteins that maintain the DNA damage response pathway are critical in preserving genomic stability. One essential DNA repair protein is the Breast Cancer Associated gene, BRCA1. BRCA1 is essential for mediating the DNA damage response, facilitating DNA damage repair, and activating key cell cycle checkpoints. Moreover, mutations in BRCA1 lead to a higher incidence of breast and ovarian cancer, highlighting the importance of BRCA1 as a tumor suppressor. In an effort to better understand how BRCA1 carried out these functions, researchers sought to identify additional BRCA1 interacting proteins. This led to the identification of several proteins including the BRCA1 Associated C-terminal Helicase, BACH1. Due to the direct interaction of BACH1 with a region of BRCA1 essential for DNA repair and tumor suppression, it was speculated that BACH1 may help support these BRCA1 function(s). In fact, initial genetic screenings confirmed that mutations in BACH1 correlated not only with hereditary breast cancer, but also with defects in DNA damage repair processes. The initial correlation between BACH1 and cancer predisposition was further confirmed when mutations in BACH1 were identified in the cancer syndrome Fanconi anemia (FA) (complementation group FA-J), thus giving BACH1 its new name FANCJ. These findings supported a previously established link between the FA and BRCA pathways and between FA and DNA repair. In particular, we demonstrated that similar to other FA/BRCA proteins, suppression of FANCJ lead to a substantial decrease in homologous recombination and enhanced both the cellular sensitivity to DNA interstrand cross-linking agents and chromosomal instability. What remained unknown was specifically how FANCJ functioned and whether these functions were dependent on its interaction with BRCA1 or other associated partners. In fact, we identified that FANCJ interacted directly with the MMR protein MLH1. Moreover, we found that the FANCJ/BRCA1 interaction was not required to correct the cellular defects in FA-J cells, but rather that the FANCJ/MLH1 interaction was required. Although both the FA/BRCA and MMR pathways undoubtedly mediate the DNA damage response, there was no evidence to suggest that these pathways were linked, until recently. Our findings not only indicate a physical link between these pathways by protein-protein interaction, but also demonstrated a functional link.

The DNA damage response (DDR) pathway is a complicated network of interacting proteins that function to sense and remove DNA damage. Upon exposure to DNA damage, a signaling cascade is generated. The damage is either removed, restoring the original genetic sequence, or apoptosis is activated. In the absence of DDR, cells are unable to effectively process DNA damage. Unprocessed DNA damage can lead to chromosomal changes, gene mutations, and malignant transformation. Thus, the proteins involved in DDR are critical for maintaining genomic stability. One essential DDR protein is the BRCA1 Associated C-terminal Helicase, BACH1. BACH1 was initially identified through its direct association with the BRCT domain of the Breast Cancer Associated Gene, BRCA1. Similar to BRCA1, germline mutations in BACH1were identified in patients with early onset breast cancer. Interestingly, the disease-associated mutations in BACH1 were shown to have altered helicase activity in vitro, providing a direct link between BACH1 helicase activity and disease development. The correlation between BACH1 and cancer predisposition was further confirmed by the identification of BACH1 as the cancer syndrome Fanconi anemia (FA) gene product, FANCJ. Similar to other FA proteins, suppression of FANCJ leads to decreased homologous recombination, enhanced sensitivity to DNA interstrand crosslinking (ICL) agents, and chromosomal instability. In an effort to further understand the function of FANCJ in DDR, FANCJ was shown to directly associate with the mismatch repair (MMR) protein MLH1. This interaction is facilitated by lysines 141 and 142 within the helicase domain of FANCJ. Importantly, the FANCJ/MLH1 interaction is critical for ICL repair. Furthermore, in an attempt to dissect the binding site of FANCJ on MLH1, we discovered an HNPCC associated MLH1 mutation (L607H) that has intact mismatch repair, but lacks FANCJ interaction. In contrast to the MLH1 interaction, the FANCJ/BRCA1 interaction was not required for correcting the cellular defects in FANCJ null cells. Thus, in an effort to understand the functional significance of the FANCJ/BRCA1 interaction, we discovered that FANCJ promotes Pol η dependent translesion synthesis (TLS) bypass when uncoupled from BRCA1. In this thesis, we provide evidence suggesting that FANCJ and MLH1 are functionally linked and that the interaction of these proteins is critical for repair choice.

No description available.

No / The Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives as well as in controlling stem cell activity. Here, we show that during murine skin morphogenesis, Lhx2 is expressed in the hair follicle (HF) buds, whereas in postnatal telogen HFs Lhx2(+) cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Remarkably, Lhx2(+) cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, as wound re-epithelization is significantly retarded in heterozygous Lhx2 knockout (+/-) mice, whereas anagen onset in the HFs located closely to the wound is accelerated compared with wild-type mice. Cell proliferation in the bulge and the number of Sox9(+) and Tcf4(+) cells in the HFs closely adjacent to the wound in Lhx2(+/-) mice are decreased in comparison with wild-type controls, whereas expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2(+/-) mice is inhibited by administration of Lgr5 siRNA. Finally, Chip-on-chip/ChIP-qPCR and reporter assay analyses identified Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells, and promotes wound re-epithelization, whereas it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as an important regulator of epithelial stem cell activity in the skin response to injury.