Tesis sobre el tema ""NAC transcription factors""

Myogenesis is a complex and tightly regulated process, the end result of which is the formation of multinucleated myofibers. Muscle formation requires the precise expression of multiple myogenic regulatory factors (MRFs), whose expression regulates transcription of muscle specific proteins. Alteration in the expression of a muscle specific gene or protein ultimately results in muscle dysfunction. The inappropriate expression of factors that control muscle development may also be a contributing factor in Rhabdomyosarcoma, a pediatric cancer that accounts for most soft tissue sarcomas that arise in children. Previous studies suggest that the regulation of αNAC, BTF3, and skNAC are vital for normal myogenesis. Alterations in these factors results in retarded development and severe disorganization of muscle tissue. We hypothesized that αNAC, BTF3, and skNAC are imperative transcription factors whose dysregulation significantly alters the kinetics of C2C12 cell (murine skeletal muscle cells) myogenesis. We have shown that erroneous expression of these transcription factors is detrimental to myogenesis. In addition, we have shown that these transcription factors are recruited to muscle specific gene promoters during the myogenic differentiation program and the expression of αNAC, BTF3, and skNAC may potentiate the expression of the MRFs. Together, our experiments suggest that the expression of αNAC, BTF3, and skNAC are essential for the normal progression of myogenesis.

Wheat is the most important grain crop grown in our country providing greatest part of the daily nutritional requirement. Abiotic factors including salinity, drought, cold and heat stresses affect quality and yield of wheat varieties used for the production of both bread and pasta flour. NAC proteins form one of the widest families of plant specific transcription factors. Members of this family are related with development, defense and abiotic stress responses. TaNAC69-1 and TtNAM-B2 genes were isolated from T.aestivum and T.turgidum, respectively. Then they were cloned into different monocot and dicot expression vectors to be used for further wheat and tobacco genetic transformation studies. To understand effects of salinity, drought, cold and heat stresses on expression profiles of TaNAC69-1 and TtNAM-B2 genes, quantitative real time PCR was performed. The time series expression profiles of TaNAC69-1 show that it was signi

THESIS OUTLINE In the present thesis work, the senescence process in fruit and leaf has been investigated through the analysis of different mutant lines of Arabidopsis thaliana and Solanum lycopersicum. In particular, the work has focused on the downregulation of three NAC transcription factor genes, NAC058 (AT3G18400), NAC100 (AT5G61430, both analysed in Arabidopsis), and HEBE (Solyc12g036480), analysed in tomato. All of them have been selected for the analysis due to their putative role in senescence, e.g. their expression has been detected in senescing siliques (NAC058, NAC100) and in ripening berries (NAC100). In addition, HEBE is the putative ortholog in tomato of NAC058 of Arabidopsis. The work has been divided in the following chapters: - CHARACTERIZATION OF NAC058, A NEGATIVE SENESCENCE REGULATOR IN SILIQUES OF Arabidopsis thaliana - in this section, NAC058 is identified as fruit-specific negative regulator of senescence. Overexpression lines are analysed and NAC058 expression is localized in different tissues of 21 the siliques. Moreover, the bond between this transcription factor and phytohormones is preliminarly evaluated. - NAC100, A NOVEL NAC TRANSCRIPTION FACTOR THAT NEGATIVELY REGULATES FRUIT SENESCENCE IN Arabidopsis thaliana – in this chapter, a comparison between transcriptomic data of Arabidopsis senescing siliques and tomato ripening berries allows to identify conserved NAC genes expressed during fruit maturation. 7 Arabidopsis lines carrying T-DNA insertions in these genes are analysed looking at senescence-related traits and, among them, nac100 is selected as putative negative regulator of silique senescence. In addition, NAC100 is found to affect silique development. - HEBE, A NOVEL POSITIVE REGULATOR OF LEAF SENESCENCE IN Solanum lycopersicum – in this section, HEBE, the putative ortholog of NAC058 in tomato, is temporary silenced in tomato plants through Virus-Induced Gene Silencing (VIGS), resulting in a stay-green phenotype. The work has been published in 2020 (paper in attachment at the chapter).

Le blé tendre, Triticum aestivum, est une des céréales les plus cultivées dans le monde. Le changement climatique qui se développe actuellement contraint fortement les cultures et altère leur rendement. La compréhension des mécanismes de réponse du blé tendre aux stress abiotiques est donc une problématique d’actualité. Plusieurs grandes familles de facteurs de transcription, dont la famille NAC,interviennent dans le développement de la plante et dans sa réponse aux stress environnementaux. Cette thèse, structurée en 3 volets, est ciblée sur l’étude de la famille NAC chez le blé tendre : les TaNAC. Dans un premier temps, nous avons étudié la structuration génomique et phylogénétique des 488 membres de la famille TaNAC, recensés à partir de la base de données la plus récente du blé tendre.Nous avons aussi étudié l’histoire évolutive de cette famille, qui a été marquée par des événements de duplication et de rétroposition. Enfin, une analyse de sa diversité allélique a permis d’identifier des gènes qui présentent des SNP montrant une forte association avec des paramètres d’accumulation des protéines de réserve dans le grain. Le deuxième chapitre de cette thèse a porté sur l’étude de l’expression de ces 488 gènes TaNAC dans plusieurs organes et en réponse aux stress thermique et sécheresse. Une analyse globale a été réalisée à partir de données bio-informatiques, suivie d’une étude in planta de l’expression d’une sélection de 23 gènes. Les profils d’expression obtenus ont révélé l’existence de 4 gènes TaNAC, encore jamais décrits dans la littérature et qui interviennent dans le développement du grain de blé tendre mais aussi dans sa réponse adaptative à plusieurs stress abiotiques. Le troisième volet de cette thèse a donc porté sur la caractérisation génétique, moléculaire et physiologique de ces 4 facteurs de transcription TaNAC. Ils appartiennent à un clade rassemblant des séquences présentant des similitudes génomique et structurale. De plus, ils sont localisés dans le noyau et leurs profils d’expression sont similaires, avec toutefois un niveau variable entre gènes et entre homéologues pour chaque gène. En réponse à un stress thermique modéré, ce profil d’expression est accéléré au cours du développement du grain ; le stade 120°Cj étant le stade clé qui montre la plus grande différence d’expression de ces gènes entre les conditions contrôle et stressée. Pour des raisons techniques, la production de plantes transgéniques sur- et sous-exprimant ces gènes n’a pas permis de valider l’implication de ces 4 TaNAC dans le développement du grain et en réponse à la température. Une analyse de génétique d’association a toutefois permis de mettre en évidence un lien entre des marqueurs moléculaires situés dans ces gènes et l’accumulation des protéines de réserve.Globalement, les résultats obtenus ont montré que des membres de la famille TaNAC sont impliqués dans le développement du blé tendre et dans sa réponse aux stress abiotiques. Plus particulièrement, 4 facteurs de transcription TaNAC semblent jouer un rôle clé dans l’accumulation des protéines dans le grain en réponse à un stress thermique modéré
Bread wheat, Triticum aestivum, is one of the most cultivated cereal in the world. The climate change that is currently developing strongly constrains crops and impairs their yield. Understanding the wheat response mechanisms to abiotic stresses is therefore a current issue. Several major families of transcription factors, including the NAC family, are involved in the plant development and its response to environmental stresses. This thesis, structured in three parts, is focused on the study of the NAC family in bread wheat (TaNAC).First, we studied the genomic and phylogenetic structure of the 488 members of the TaNAC family identified from the latest database of bread wheat. We also studied the evolutionary history of this family, which was marked by duplication and retroposition events. Finally, an analysis of its allelic diversity allows us to identify genes with SNP showing a strong association with storage protein accumulation parameters in the grain. In a second part, we studied the expression of these 488 TaNAC genes in several organs and in response to heat and drought. An overall analysis was performed using bioinformatic data, followed by an in planta study of the expression of a selection of 23 genes. The expression profiles revealed that four TaNAC genes, never described in the literature, are involved in the wheat grain development but also in its adaptive response to several abiotic stresses. In a third part, we focused on the genetic, molecular and physiological characterization of these four TaNAC transcription factors. They belong to a clade gathering sequences with genomic and structural similarities. Moreover, they are localized in the nucleus and their expression profiles are similar, with a variable level between genes and between homeologs for each gene. In response to moderate heat stress, this expression profile is accelerated during grain development and a key stage at 120°Cj was identified, it shows the greatest difference in genes expression level between control and stressed conditions. For technical reasons, the production of transgenic plants over- and under-expressing these genes did not validate the involvement of these 4 TaNAC in grain development and in its temperature response. An association genetic analysis, however, showed a link between molecular markers located in these genes and the storage proteins accumulation. Overall, the results showed that members of the TaNAC family are involved in the bread wheat development and its response to abiotic stresses. In particular, four TaNAC transcription factors appear to play a key role in grain protein accumulation in response to a moderate heat stress

Wood has a wide variety of uses and is arguably the most important renewable raw material. The composition of xylem cell types in wood determines the utility of different types of wood for distinct commercial applications. Using expression profiling and phylogenetic analysis, we identified many xylem-associated regulatory genes that may control the differentiation of cells involved in wood formation in Arabidopsis and poplar. Prominent among these are NAC-domain transcription factors (NACs). In addition to their roles as regulators of xylem differentiation, NACs are regulators of meristem development, organ elongation and separation. We studied a subset of Populus and Arabidopsis NACs with putative involvement in xylem cell expansion and elongation (XND1/ANAC104, PopNAC118, PopNAC122, PopNAC128, PopNAC129), and secondary cell wall synthesis (ANAC073, PopNAC105, PopNAC154, PopNAC156, PopNAC157). Using quantitative Real-Time PCR, we evaluated expression of the selected Populus NACs in a developmental gradient and in response to bending stress. We prepared transgenic Arabidopsis and Populus plants with increased or decreased expression of select NAC genes. For dominant repression of target gene expression, we evaluated transgenic plants expressing translational fusions of NAC-EAR (ERF amphiphilic repressor) chimeras through chimeric repressor silencing-technology (CRES-T). XND1 overexpression in Populus and Arabidopsis resulted in severe stunting and suppression of xylem differentiation. Overexpression of PopNAC122, an XND1 ortholog, yielded an analogous phenotype in Arabidopsis. Populus XND1 overexpressors lacked phloem fibers and showed a reduction in cell size and number, vessel number and frequency of rays. Knowledge gained through characterization of these wood-associated regulatory genes can be used to optimize molecular breeding and genetic engineering strategies for improved wood quality and increased biomass.
Master of Science

Wood formation is heavily exploited for the manufacturing of pulp, paper, sustainable biomaterials and, potentially, biofuels. Eucalyptus is a favourable fast-growing, short rotation plantation crop grown over millions of hectares globally for its superior fiber properties. Understanding the molecular biology of secondary cell wall (SCW) formation in trees, and in particular how it is transcriptionally and epigenetically regulated, lays the foundation for enhanced woody trait improvement strategies in tree biotechnology. Transcriptional networks regulating SCW biosynthesis have been discovered in the herbaceous model plant Arabidopsis thaliana, in which NAC domain transcription factors (TFs) play a prominent role. The functions of many NAC domain TFs remain to be resolved, and their regulatory roles and evolution in Eucalyptus is unknown. Functional genomics studies of Eucalyptus TFs are currently challenged by a lack of established high-throughput genomics techniques commonly applied to model organisms. In this study, we aimed to better understand NAC family evolution and the epigenetic regulation of xylogenesis in E. grandis, and characterize the role of NAC domain TF SND2 in transcriptional regulation of SCW biosynthesis in A. thaliana and E. grandis. Comparative genomics and bioinformatics analyses of 189 curated gene models of the E. grandis NAC family, one of the largest described to date, revealed extensive tandem duplication in stress response-associated subfamilies, while SCW-associated subfamilies were generally conserved among angiosperms. Novel candidates for wood and tension wood formation as well as cold-stress tolerance were identified from transcriptional profiling in E. globulus and E. grandis. We identified the phenotypic effects and putative targets of the NAC domain TF SND2 in A. thaliana using microarray, microscopy and cell wall chemistry analyses. Moderate SND2 overexpression upregulated genes involved in cellulose, xylan, mannan, signaling and lignin polymerization processes and affected mannose, rhamnose and lignin components of stem cell walls, while strong overexpression resulted in reduced interfascicular fiber SCW deposition. SND2 overexpression in Eucalyptus somatic xylem sectors increased cross-sectional fiber cell area. We optimized a chromatin immunoprecipitation sequencing (ChIP-seq) approach and applied it to developing secondary xylem of mature E. grandis trees to identify the targets of the E. grandis ortholog of SND2, EgrNAC170. In validating the approach, we addressed the regulatory role of the epigenetic mark trimethylated lysine 4 of histone H3 (H3K4me3) in this tissue, showing a strong association with expressed loci, occupation of regions close to transcriptional start sites and tight correlation with transcript abundance, especially that of broadly expressed genes but also genes associated with SCW formation. A pilot study of EgrNAC170 targets was performed using the high-throughput ChIP-seq approach, identifying over 3,000 putative targets in E. grandis developing secondary xylem, but showing evidence that further ChIP-seq data are required for reliable target identification. The results of this thesis contribute to science an understanding of the unique evolution of NAC proteins in Eucalyptus, knowledge of the function of SND2/EgrNAC170 as possible candidates for tree biotechnology, the first genomic profile of a histone modification in developing wood and a high-throughput ChIP-seq protocol for the study of native protein-DNA interactions in developing xylem.
Thesis (PhD)--University of Pretoria, 2014.
Genetics
PhD
Unrestricted

The NAM-B1 transcription factor increases grain protein content, alters grain micronutrient content and accelerates monocarpic senescence, often without imposing a yield penalty. The aim of this thesis was to understand the mechanisms by which NAM-B1 influences nutrient remobilisation and monocarpic senescence to cause these effects. To achieve this I have examined the expression patterns of NAM-B1 and its homologues during development. I have studied the effects of NAM-B1 on nutrient transport, photosynthetic capacity and grain filling using a range of molecular biology and physiological techniques. Finally to understand the network of genes which NAM-B1 regulates I have used chromatin-immunoprecipitation followed by next-generation sequencing (ChIP-seq) to identify downstream targets, and compared these to differentially expressed genes in plants with down-regulated expression of NAM-B1 homologues (NAM RNAi plants). I have found that NAM-B1 expression increases after anthesis in both vegetative and reproductive tissues, including the grain. In stem and leaf tissues I identified that NAM genes are highly expressed in the vascular bundles, which might be important for nutrient transport. However I did not find evidence for NAM genes altering xylem or phloem transport. I found that in NAM RNAi plants, grain development was decoupled from flag leaf senescence. In RNAi plants starch synthesis enzymes were less active during the middle of grain filling than in control plants, potentially resulting in the reallocation of photosynthate to the stems as water soluble carbohydrates. Many of the putative NAM-B1 target genes identified by ChIP-seq have functions related to photosynthesis and validation of these candidate genes is ongoing. In summary I have identified putative NAM-B1 target genes and found that NAM-B1 may act in a tissue specific manner to regulate monocarpic senescence and grain filling. Furthermore I have highlighted novel functions related to carbohydrate metabolism in stems and the grain.

Les protéines TCP constituent une famille de facteurs de transcription spécifiques des végétaux impliqués dans la régulation de la morphologie des plantes. Ce travail a pour objectif d'étudier l'évolution et la fonction de ces protéines. Dans la première partie, une étude structurelle et évolutive de la famille TCP permet de dater l'apparition de la famille avant la divergence du groupe d'algues des Zygnémophytes, il y a entre 650 et 800 millions d'années et indique une expansion permanente de la famille TCP au cours de l'évolution des Phragmoplastophytes. Dans la seconde partie, une analyse fonctionnelle in planta du gène AtTCP9 d'Arabidopsis a été entreprise. Le gène rapporteur de la glucuronidase (GUS) en fusion transcriptionnelle avec le promoteur d'AtTCP9 s'exprime tout au long du développement de la plante, majoritairement dans les vaisseaux. La protéine AtTCP9 est détectée par western dans les feuilles et les fleurs d'Arabidopsis mais pas dans les siliques. La caractérisation de mutants d'insertion ainsi que l'analyse de plantes transformées avec des fusions AtTCP9-domaine répresseur (EAR) ou activateur (VP16) sous contrôle d'un promoteur inductible (XVE) n'ont pas permis d'identifier de fonction mais suggèrent l'intervention d'une régulation post-transcriptionnelle de l'expression de AtTCP9. Un ARN antisens naturel (cis-NAT: cis-Natural Antisense Transcript) codé au même locus qu'AtTCP9 ainsi qu'un siRNA (small interfering RNA) correspondant à ce locus ont été caractérisés. Les résultats préliminaires semblent indiquer une possible régulation par inhibition traductionnelle d'AtTCP9 dans les siliques ainsi que l'existence d'un mécanisme de régulation inhabituel par un NAT et un siRNA
TCP proteins are plant-specific transcription factors involved in plant morphogenesis control. The objective of this work was to determine the evolution and the function of the TCP family transcription factors. In the first part, we made a structural and evolutionary study of the TCP family that indicated appearance of the family before the divergence of Charophytes, 650 to 800 mya. Phylogenetics indicated permanent expansion of the family during the evolution of the Phragmoplastophytes. In the second part, we undertook a functional study of a TCP Arabidopsis thaliana gene (AtTCP9). Plants bearing transcriptional fusion between AtTCP9 promoter and GUS reporter gene revealed an expression during the plant growth, especially in the vasculature. AtTCP9 protein was detected by western in leaves and flowers but not in siliques. Analysis of insertion mutant lines and plants bearing chimeric fusions between AtTCP9 and a repressor (EAR) or an activator (VP16) domain, under control of an inducible promoter (XVE) didn't allow us to identify the function of this gene, but the results suggest the involvement of a post-transcriptional regulation on AtTCP9. A Natural Antisense Transcript expressed at the same locus than AtTCP9 (cis-NAT) and an associated small interfering RNA (siRNA) were characterised. Preliminary results seem to indicate a translational inhibition of AtTCP9 in siliques and an unusual mechanism of regulation involving a NAT and a siRNA

La adaptación de la levadura Saccharomyces cerevisiae a condiciones de alta osmolaridad está mediada por la vía de HOG ((high-osmolarity glycerol). La activación de esta vía induce una serie de respuestas que van a permitir la supervivencia celular en respuesta a estrés. La regulación génica constituye una respuesta clave para dicha supervivencia. Se han descrito cinco factores de transcripción regulados por Hog1 en respuesta a estrés osmótico. Sin embargo, éstos no pueden explicar la totalidad de los genes regulados por la MAPK Hog1. En el presente trabajo describimos cómo el complejo transcripcional formado por las proteínas Rtg1 y Rtg3 regula, a través de la quinasa Hog1, la expresión de un conjunto específico de genes. Hog1 fosforila Rtg1 y Rtg3, aunque ninguna de estas fosforilaciones son esenciales para regulación transcripcional en respuesta a estrés. Este trabajo también muestra cómo la deleción de proteínas RTG provoca osmosensibilidad celular, lo que indica que la integridad de la vía de RTG es esencial para la supervivencia celular frente a un estrés osmótico.
In Saccharomyces cerevisiae the adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway, which elicits different cellular responses required for cell survival upon osmostress. Regulation of gene expression is a major adaptative response required for cell survival in response to osmotic stress. At least five transcription factors have been reported to be controlled by the Hog1 MAPK. However, they cannot account for the regulation of all of the genes under the control of the Hog1 MAPK. Here we show that the Rtg1/3 transcriptional complex regulates the expression of specific genes upon osmostress in a Hog1-dependent manner. Hog1 phosphorylates both Rtg1 and Rtg3 proteins. However, none of these phosphorylations are essential for the transcriptional regulation upon osmostress. Here we also show that the deletion of RTG proteins leads to osmosensitivity at high osmolarity, suggesting that the RTG-pathway integrity is essential for cell survival upon stress.

Introdução: A hipóxia tumoral tem papel de fator prognóstico independente, estando relacionado a fenótipo mais agressivo, com maior capacidade de proliferação, invasão e disseminação metastática, além de maior resistência ao tratamento sistêmico e à radioterapia. As alterações do microambiente decorrente do baixo nível de oxigênio pode levar a formação de células tronco tumorais (CTTs) em células de câncer de mama, que são identificadas através da expressão de aldeído desidrogenase (ALDH1A1). Modelos experimentais para estudo da influência da hipóxia no desenvolvimento e progressão do câncer demonstram dados inconclusivos e controversos e estão limitados a modelos in vitro e modelos in vivo que submetem os camundongos a estado de hipóxia sistêmica. Em nosso estudo, desenvolvemos um modelo in vivo para estudar se o estado de hipóxia controlado, direcionado ao local do tumor, pode induzir a formação de CTT em câncer de mama de camundongos. Métodos: células da linhagem 67 NR foram introduzidas dentro do córtex renal de fêmeas de camundongos BALB/c. Foi colocado um clipe de aneurisma no hilo renal por 60 minutos para provocar hipóxia direcionada ao local do tumor. Os camundongos foram sacrificados após 24 horas ou 7 dias da hipóxia e foi realizada análise histológica para confirmar as mudanças induzidas pela isquemia na córtex renal e tumoral. Realizada dissecção tumoral microscópica e RT-PCR para análise da expressão CA9, HIF1A, HIF2A, Slug, SOX 9 e ALDHA1(TaqMan® Gene Expression Assay) no grupo controle e da hipóxia. TBP e HPRT foram os genes housekeeping e o crescimento in vitro de linhagem de células 67NR, usado como referência. Resultados: Todos os camundongos enxertados com células 67NR no cortex renal desenvolveram tumores locais. Foi observada necrose tubular aguda (NTA), decorrente da hipóxia tecidual direcionada. Os genes foram diferentemente expressos, comparando com as linhagens celulares. Notamos aumento significativo da ativação da via da hipóxia por aumento da expressão gênica de CA9, HIF1A e HIF2A. Ocorreu uma modulação para formação de CTTs. Obervamos aumento de 2,9 e de 5,7 respectivamente, na expressão de SLUG, relação SLUG/SOX9 após 24 horas de hipóxia, redução de 2,7 vezes na expressão de SOX9 e não houve aumento na expressão de ALDH1. Entretanto, houve aumento de 2,1 vezes na expressão de ALDH1 7 dias após a hipóxia. Conclusão: Houve aumento da expressão de Slug e Slug/SOX9 precocemente ao aumento da expressão de ALDH1, em ambiente de hipóxia tumoral, o que sugere que há probabilidade de participação de Slug e SOX9 na formação de CTTs
The tumoral hypoxia is an independent prognostic factor in many solid malignat tumors. Low oxygen tension has been related to more aggressive phenotype, greater proliferation capacity, increased invasiveness and metastatic dissemination, and resistance to systemic treatment and radiotherapy. Changes in the microenvironment due low oxygen level lead to cancer stem cells formation (CSC) in breast câncer. The aldehyde dehydrogenase (ALDH1) cellular expression has been decribed as a CSC marker. Experimental models to study the influence of hypoxia on the development and progression of cancer have shown inconclusive and controversial data and are restricted to models in vitro and in vivo models in mice undergoing a state of systemic hypoxia. In our study, we developed an in vivo model to study whether the state of hypoxia controlled targeted to the tumor site may induce the formation of CSC in breast cancer in mice. Methods: 67 NR lineage cells were engrafted into the renal cortex of female BALB / c mice. Aneurysm clip was placed in the renal hilum for 60 minutes to induce hypoxia targeted to the tumor site. Mice were sacrificed after 24 hours or 7 days of hypoxia, and histological analysis was performed to confirm ischemia-induced renal cortex changes. Tumors were microdissected and RT-PCR was carried out to analyze the expression of CA9, HIF1A, HIF2A, Slug, Sox 9 and ALDHA1 genes (TaqMan® Gene Expression Assay) in the control and hypoxia groups. TBP and HPRT were used as housekeeping genes and the cell lines 67NR gowing in culture plate was used as reference. Results: All mice engrafted with cells in the renal cortex 67NR developed local tumors. Acute tubular necrosis (ATN) was observed, resulting from the targeted tissue hypoxia. The genes were expressed differently, compared with cell lines. We notice a significant increase in activation of the hypoxia by increased gene expression of CA9, HIF1A and HIF2A, 24 hours after renal tumor ischemia. There was increased by 2.9 and 5.7 fold, respectively, in the expression of SLUG and SLUG/SOX9, no difference in ALDH1 and reduced by 2.7 fold in the expression of SOX 9, 24 hours after ischemia.There was increased by 2.1 fold in ALDH1 expression 7 days after ischemia. Conclusion: There was increased in expression of SLUG and SLUG/SOX9 early expression of ALDH1 in tumor hypoxia environment. This suggests are likely SLUG and SOX9 have role in cancer stem cell transformation

Orientador: Claudio Alexandre Gobatto
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Educação Física
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Resumo: Sabe-se que o estresse físico exerce uma função moduladora na expressão gênica dos MCTs 1 e 4 por meio de vias de sinalização moleculares aparentemente distintas envolvendo o co-ativador-1 'alfa' do receptor gama ativado por proliferador do peroxissomo (PGC-1?) e subunidade 1 'alfa' do fator induzível por hipóxia (HIF-1?) respectivamente. Apenas uma única sessão de exercício de resistência (endurance) está associada ao aumento na expressão do MCT1 e PGC-1?, mas não do MCT4, no músculo esquelético vasto lateral de humanos, enquanto o exercício intermitente de alta intensidade parece afetar ambos MCTs 1 e 4 além do PGC-1?. No entanto pouco se conhece sobre o efeito simultâneo do estresse físico sobre o HIF-1?, MCts 1 e 4 e PGC-1? em diferentes tecidos e tipos de fibra. É provável que tanto a expressão quanto a transcrição dos co-ativadores e fatores de transcrição envolvidos na modulação dos MCTs 1 e 4 frente ao estresse físico sejam afetadas pelas características da atividade e ainda variem de acordo com o tipo e especificidade do tecido analisado. Dessa forma, o objetivo desse estudo foi verificar o efeito agudo de uma única sessão de natação até exaustão ou de 30 minutos contínuos ou 25 minutos acumulados intermitentemente, em uma intensidade equivalente ou 20% superior a máxima fase estável de lactato, sobre a expressão gênica e conteúdo protéico dos HIF-1?, MCTs 1 e 4, PGC-1?, imediatamente, 2, 4 e 8 horas após a sessão de exercício, em tecidos chaves para metabolismo do lactato (fibras esqueléticas I e II, fígado, coração) de ratos. O exercício físico aumenta a expressão proteica e mRNA em relação ao grupo controle para maior parte dos genes que foram analisados, porém, não há diferenças entre os grupos exercitados independente do tecido e do protocolo utilizado. Com exceção do tecido hepático cuja apenas a expressão de PGC-1? mRNA é estimulada, uma única sessão de exercício induz diferentes respostas ao longo de 8 horas na expressão mRNA e conteúdo de HIF-1?, MCTs 1 e 4, PGC-1?. Uma sessão contínua de volume reduzido ou uma sessão intermitente em intensidade 20% superior a MFEL, resultam nas mesmas adaptações de uma sessão contínua de 30 minutos de duração em intensidade equivalente a MFEL
Abstract: It is known that physical stress plays a role on regulating the gene expression of MCTs 1 and 4 by distinct molecular signaling pathways involving the peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1?) and hypoxia inducible factor 1 alpha subunit (HIF-1?) respectively. Only a single endurance session is associated with increased expression of both PGC-1? and MCT1, but not of the MCT4 in the muscle vastus lateralis of humans, while the intermittent exercise of high intensity seems to affect, besides the PGC-1?, both MCTs 1 and 4. However, the knowledge about the simultaneously effect of the exercise stress on the HIF-1?, MCTs 1 and 4 and PGC-1? in different types of tissues and skeletal muscles is unknow. Probably, the transcription factors and the coativators involved in the exercise induced modulation of MCTs 1 and 4 can being differently affected by the exercise intensity and may vary according to the type and metabolic specificity of the tissue. Thus, the aim of this study was to investigate the acute effect of a single swimming session of 30 continuous minutes or 25 minutes accumulated intermittently or until exhaustion in the intensity equivalent or 20% higher than the maximum lactate steady state (MLSS), on the gene expression and protein content of HIF-1?, MCTs 1 and 4, PGC-1?, immediately, 2, 4 and 8 hours after, in key tissues to the lactate metabolism (skeletal muscle of type I and II, liver, heart) of rats. Physical exercise increased protein content and mRNA expression for most of the analyzed genes, however, there are no differences between the exercised groups independently of the tissue or protocol used. With the exception to liver, where only PGC-1? mRNA was stimulated, a single exercise bout induced different responses throughout 8 hours on mRNA expression and content of HIF-1?, MCTs 1 and 4, PGC-1?. Both, continuous or intermittent exercise, of reduced volume and in higher intensity (20%) results in similar responses of a continuous session of 30 minutes duration in the MLSS intensity
Doutorado
Biodinamica do Movimento e Esporte
Doutor em Educação Física

As mutações no gene PROP1 são a causa genética mais comum da deficiência combinada de hormônios hipofisários. Até o momento, diversas mutações missense e pequenas deleções foram descritas sendo a mutação 301-302 delAG a mais freqüente. Nosso objetivo foi estudar as mutações em DNA de pacientes com hipopituitarismo e padronizar a extração de DNA de células de swab oral, usando um método com NaCl e comparar com um kit comercial (Purigene, Minneapolis, EUA). Amplificamos os 3 exons do gene PROP1 do DNA obtido de células orais e de sangue periférico. Identificamos a mutação 301-302delAG em 6 pacientes, 4 em homozigose (33%) e 2 em heterozigose (16%) e a mutação G51A em heterozigose em um único paciente. Em dois irmãos, filhos de pais consangüíneos, não foi possível amplificar os 3 exons do gene PROP1 enquanto que os os genes LHX3 e LHX4 foram amplificados com sucesso. Para confirmar a hipótese de deleção do PROP1, o Southern blotting foi realizado usando como sonda o produto de PCR do exon 2 do gene PROP1 e um fragmento do gene CYP21A2 como sonda controle. A banda referente ao CYP21A2 estava presente nos pacientes e nos controles enquanto a banda referente ao PROP1 estava ausente nos irmãos e presente na mãe e nos controles. Para definir a extensão da deleção usamos um mapa de STS próximos ao gene e o STS GDB:314805 localizado a 6,0 kb a montante do PROP1 não foi amplificado nos pacientes. Entretanto, o gene Q8N6H0 a 18 kb a juzante e o STS WI-16216 a 59 kb a montante do PROP1 foram amplificados com sucesso nos pacientes e controles indicando que a deleção está localizada dentro de 81 Kb. Para determinar os limites da deleção, várias reações de PCR foram realizadas com primers desenhados progressivamente distantes de gene PROP1, cobrindo toda a região. Isto nos permitiu determinar a região deletada de 9,6 kb a juzante e 11 kb a montante do gene PROP1, com o tamanho máximo deletado de 18,4 kb. Por ambos os métodos de extração obtivemos um DNA de boa qualidade, permitindo o amplificação dos 3 exons do gene PROP1. A extração com NaCl foi mais rápida e mais barata resultando em maior quantidade de DNA quando comparada com o kit comercial. Em conclusão, descrevemos a deleção completa do gene PROP1 em dois irmãos com o fenótipo clássico de hipopituitarismo associado à hipófise hipoplásica ou aumentada e padronizamos a extração de DNA de células de mucosa oral com NaCl, que apresentou custo mais baixo e resultado mais rápido quando comparado a extração por um kit comercial, indicando que o swab oral é uma fonte prática de obtenção de DNA para estudos genéticos.
PROP1 gene mutations are the most common cause of genetic combined pituitary hormone deficiency. To date, several missense mutations and small deletions have been described and the 301-302 del AG is the most frequent. Our objective was to study PROP1 mutations in patients with hypopituitarism and standardize DNA extraction from an oral swab, using the NaCl method, comparing it with a commercial kit (Purigene, Minneapolis, USA). We amplified the 3 exons of PROP1 gene in DNA obtained from oral cells and peripheral blood cells. We identified the delAG301-302 mutation in 6 patients, 4 in homozygous (33%) and 2 in heterozygous (16%) state and G51A mutation in heterozygous state in a single patient. In two siblings, a boy and a girl, born to consanguineous parents we failed to amplify PROP1 gene by PCR whereas LHX3 and LHX4 genes were successfully amplified. To confirm the hypothesis of PROP1 gene deletion, Southern blotting was performed using PROP1 exon 2 gene PCR product as a probe and a fragment of CYP21A2 gene as a control probe. The CYP21A2 band was present in patients and controls whereas PROP1 band was absent in both siblings and present in their mother and in controls. To define the extension of this deletion we used STS mapping approach and no amplification for a STS GDB:314805 6.0 kb downstream of PROP1 was found. However, Q8N6H0 gene located 18 kb upstream and the STS WI-16216 located 59 kb downstream of PROP1 were successfully amplified indicating that the deletion is placed within 81 Kb. To determine the limits of the deletion a number of PCR covering this region were then carried out with primers located progressively distant from PROP1. This allowed us determine the deleted region from 9.6 kb upstream to 11 kb downstream of PROP with a maximum deletion size of 18.4 kb. Both methods yielded good quality DNA, allowing the amplification of 3 exons of PROP1 gene. The NaCl method showed to be faster and less expensive, resulting in a larger amount of DNA when compared with the commercial kit. In conclusion, we describe a complete deletion of PROP1 gene in two siblings with classical hypopituitarism phenotype associated with hypoplastic or enlarged pituitary gland and standardized the DNA extraction of oral cells with NaCl, which presented lower costs and faster results, when compared with the extraction by a commercial kit indicating that oral swabs are a reliable DNA source for genetic studies.

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
Periodontal disease is an inflammatory condition of infectious nature characterized by destruction of protecting and supporting dental tissues. It happens as a response produced by the host when attacked by microorganisms. Several factors are involved in this process. Among them, cytokines are key regulatory molecules in this immune response, playing a role either protective and/or destructive in lesion progression. Thus, this study investigated the immunohistochemical expression of IFN- , GATA-3, IL-17, IL-23, IL-6 and TGF- in gingival tissues of humans, in an attempt to gain a better understanding of the participation of Th1, Th2 and Th17 immune responses in the development of periodontal disease processes. To this end, eighty-two samples of gingival tissues were divided into three groups: Group 1 = 15 (samples of healthy gum tissue as controls), Group 2 = 36 (samples with chronic gingivitis) and Group 3 = 31 (samples with chronic periodontitis). All cases were submitted to morphological analysis from sections stained with hematoxylin and eosin and then subjected to staining by immunohistochemistry using the streptavidin-biotin method. Results showed positive labeling for all proteins. Nonetheless, we observed a greater expression of Th1 cytokines and Th17 cells in group 3. We found statistically significant difference between TGF- expression and the clinical condition of the samples (p=0.02). We conclude that Th1 and Th17 responses may act synergistically in the destructive process of periodontal tissue, overlapping the Th2 response that was also present in these tissues
A doen?a periodontal ? uma condi??o inflamat?ria de car?ter infeccioso, caracterizada pela destrui??o dos tecidos de prote??o e sustenta??o dent?rios, face ? resposta produzida pelo hospedeiro frente ?s agress?es sofridas pelos microrganismos. V?rios fatores est?o envolvidos nesse processo, sendo as citocinas as principais mol?culas reguladoras dessa resposta imune, desempenhando um papel protetor e/ou destrutivo na progress?o da les?o. Diante disso, este experimento investigou a express?o imuno-histoqu?mica de IFN- , GATA-3, IL-17, IL-23, IL-6 e TGF- em tecidos gengivais de humanos, na tentativa de se obter um maior entendimento da participa??o das respostas imunes Th1, Th2 e Th17 no desenvolvimento destes processos patol?gicos. Para tanto, oitenta e duas amostras de tecidos gengivais foram subdivididas em tr?s grupos: Grupo 1=15 (amostras de tecido gengival saud?vel-controle), Grupo 2=36 (amostras com gengivite cr?nica) e Grupo 3=31 (amostras com periodontite cr?nica). Todos os casos foram submetidos ? an?lise morfol?gica a partir de cortes corados em hematoxilina e eosina e, posteriormente, submetidas ? t?cnica de colora??o pela imuno-histoqu?mica atrav?s do m?todo da Estreptoavidina-Biotina. Os resultados mostraram positividade de marca??o para todas as prote?nas, sendo observada uma maior tend?ncia de marca??o para as citocinas das respostas Th1 e Th17 no grupo 3. Diferen?a estatisticamente significativa foi verificada entre a express?o de TGF- e a condi??o cl?nica das amostras (p=0,02). Assim, podemos concluir que as respostas Th1 e Th17 podem atuar sinergicamente no processo destrutivo dos tecidos periodontais, sobrepondo-se ? resposta Th2 que tamb?m se encontrou presente nestes tecidos

En aquest treball es presenta l'evolució molecular i estudi funcional de gens localitzats a les duplicacions segmentàries de la regió 7q11.23, implicada en la Síndrome de Williams-Beuren (SWB). S'ha datat l'aparició d'aquestes duplicacions en els últims 25 milions d'anys d'evolució i s'ha proposat un model evolutiu amb reordenaments específics i mecanismes de generació. Correlacions clínico-moleculars en els pacients amb la SWB han permès determinar que l'haploinsuficiència per NCF1, un gen localitzat a les duplicacions, és un factor protector per hipertensió. S'ha proposat un model patogènic per la hipertensió, implicant l'oxidasa NAD(P)H i estrès oxidatiu, suggerint que noves estratègies terapèutiques podrien ser utilitzades. A més, s'ha caracteritzat parcialment la funció de GTF2IRD2, un altre gen de les duplicacions. GTF2IRD2 interacciona amb altres factors de transcripció relacionats, té una localització subcel·lular variable i no s'uneix a ADN. Aquests resultats contribueixen a conèixer millor els mecanismes mutacionals i patogènics de la SWB.
This work presents the molecular evolution along with the functional analysis of the genes located in the segmental duplications flanking the 7q11.23 region, involved in Williams-Beuren syndrome (WBS). The generation of the segmental duplications has been dated to the last 25 million years of evolution and an evolutionary model with specific rearrangements and mechanisms has been proposed. Clinical-molecular correlations in WBS patients have allowed to determine that haploinsufficiency at NCF1, a gene located in the duplications, is a protective factor for hypertension. A pathogenic model for hypertension has been proposed, implicating NAD(P)H oxidase and oxidative stress, and suggesting that novel therapeutic strategies could be used. In addition, the functional characterization of another gene of the duplications, GTF2IRD2, has been partially achieved. GTF2IRD2 has been shown to interact with other related transcription factors, to display variable subcellular localization and to lack DNA binding properties. These results contribute to a better knowledge of the mutational and pathogenic mechanisms of the WBS.

Soil salinity and heat stress are two major abiotic stresses affecting plant growth and yield. Transcription factors (TFs) are key regulators in stress responses. They link stress sensing with many tolerance mechanisms by translating stress signals into changes in gene expression that ultimately contribute to stress tolerance. The NAC (NAM, ATAF and CUC) TF family have been found to be involved in responses to biotic and abiotic stresses. In this PhD project, the role of NAC TFs in response to heat and salt stress was studied in the model system Arabidopsis thaliana (Arabidopsis), and in two agriculturally relevant species, Solanum lycopersicum (tomato) and Chenopodium quinoa (quinoa). Plants have the ability to acquire thermotolerance if they are pre-exposed to a mild, non-lethal high temperature. The maintenance of acquired thermotolerance for several days is known as thermomemory. Here we investigated the role of NAC TFs in thermotolerance. The expression profiles of 104 Arabidopsis NAC TFs were measured and compared between primed and unprimed plants. Some NACs with a distinctive expression pattern in response to thermopriming were selected for further phenotypic analysis. Knock-out (KO) mutants of the ATAF1 gene showed an enhanced thermomemory phenotype compared with wild type plants (WT) and from this work, the functions of the ATAF1 gene were studied further. RNAseq co-expression analyses of ATAF1 overexpressor and ataf1 KO plants found that ANAC055 expression was co-regulated with that of ATAF1. JUBGBRUNNEN1 (JUB1) is another NAC TF involved in responses to heat, drought and salinity. In this study, the role of AtJUB1 overexpression in salinity was investigated in tomato plants. AtJUB1 overexpression resulted in higher proline levels and improved maintenance of water content and biomass in AtJUB1-overexpressing plants grown hydroponically under salinity compared with WT plants. Quinoa has recently gained much attention because of its high nutritional value and high tolerance to several stresses including drought and salinity. NAC TFs are hypothesized to play a major role in quinoa’s tolerance to abiotic stresses. In this study, the NAC TFs family were identified and investigated in the genome of quinoa. 107 NAC TF genes were identified and their transcriptional responses to different stresses including salt, drought and heat were investigated.

La vite (Vitis vinifera L.), una delle più coltivate piante da frutto, riveste notevole importanza economica in tutto il mondo. Poichè negli ultimi decenni la viticoltura sta subendo gli effetti del riscaldamento globale (Webb et al., 2007), è necessario mantenere una produzione di uva e vino di elevata qualità. Una delle maggiori sfide consiste nell’identificazione dei principali geni regolatori dello sviluppo della pianta di vite durante il ciclo vegetale annuale e, in particolare, della transizione dalla fase vegetativa a quella matura (detta véraison), durante la quale avvengono profonde modificazioni biochimiche, fisiologiche e trascrizionali. Grazie ad un'analisi di network di co-espressione sull’atlante del trascrittoma della vite e ad un dataset di dati trascrizionali di bacche (Massonnet, 2015; Palumbo et al., 2014; Fasoli et al., 2012), è stata identificata una nuova categoria di geni chiamata 'switch’; tali geni sono significativamente up-regolati durante la transizione di fase ed inversamente correlati a molti geni soppressi durante la fase matura. Tra questi, i fattori di trascrizione NAM/ATAF/CUC (NAC) rappresentano un’interessante famiglia genica dato il ruolo chiave in processi biologici come sviluppo e risposte allo stress in pianta (Jensen et al., 2014). Per la caratterizzazione funzionale cinque geni NAC sono stati selezionati come putativi principali regolatori della riprogrammazione del trascrittoma durante la maturazione della vite. VvNAC11, VvNAC13, VvNAC33 e VvNAC60 sono stati identificati come geni 'switch' dalla sopra citata analisi, mentre VvNAC03 come gene omologo a NOR (non-ripening) di pomodoro, uno dei principali regolatori della maturazione di tale frutto (Giovannoni, 2004; Giovannoni et al., 1995). I cinque NAC sono stati sovra-espressi transientemente in Vitis vinifera per ottenere una panoramica dei loro effetti primari sul trascrittoma. Sono poi state ottenute e caratterizzate dal punto di vista molecolare e fenotipico piante di vite stabilmente trasformate con VvNAC33 e VvNAC60. VvNAC33 sembra essere coinvolto nella regolazione negativa della fotosintesi poiché le foglie sovra-esprimenti tale gene contengono una minor quantità di clorofilla, mentre VvNAC60 provoca una ridotta crescita della pianta e una prematura lignificazione dello stelo rispetto ad una pianta controllo della stessa età. Questi risultati riflettono comportamenti tipici di piante in fase di maturazione e senescenza, sostenendo l’ipotesi di un ruolo fondamentale dei NAC nella transizione di fase in vite. Al fine di identificare i target che agiscono a valle dei NAC, sono state eseguite analisi microarray sulle foglie delle piante trasformate in modo transiente e stabile. In entrambe le over-espressioni è stata influenzata l’espressione di un'ampia gamma di processi cellulari tra cui, tra le categorie funzionali più rappresentate, vi sono trasporto, metabolismo secondario e attività dei fattori di trascrizione. L'identificazione di VvMYBA1, un noto regolatore della biosintesi degli anotciani in vite (Kobayashi et al., 2002), come target di VvNAC60 suggerisce un ruolo di tale NAC in processi tipici dell’inizio della maturazione. Un altro approccio utilizzato in questo lavoro è stato la complementazione funzionale del mutante nor di pomodoro con i NAC selezionati. Risultati preliminari hanno rivelato che VvNAC03 e VvNAC60 sembrano avere una funzione simile a NOR poichè riescono a maturare almeno esternamente. In conclusione, i risultati ottenuti in questo lavoro suggeriscono la capacità dei VvNAC selezionati di influenzare l'espressione di geni coinvolti nella regolazione che controlla lo sviluppo dalla fase vegeativa alla fase matura in vite. Questo lavoro ha inizato a far luce sul ruolo dei NAC nello sviluppo della vite, ma dovranno essere effettuate ulteriori analisi per ottenere una piena compresione del macchinario molecolare che regola questo complesso sistema di regolazione.
Grapevine is the most widely cultivated and economically important fruit crop in the world. Viticulture has been affected by the global warming currently under way over the past few decades (Webb et al., 2007). Improving the genetics of key grapevine functions is needed to keep producing high quality grapes and wine. In this context, a challenging task is to identify master regulators that program the development of grapevine organs and control transition from vegetative-to-mature growth featured by grape berries during the annual plant cycle. This transition, called véraison, is marked by profound biochemical, physiological and transcriptomic modifications that allow vegetative green berries to enter the ripening process. Thanks to an integrated network analysis performed on the grapevine global gene expression atlas and from a large berry transcriptomic data set (Massonnet, 2015; Palumbo et al., 2014; Fasoli et al., 2012) a new category of genes, called ‘switch’ genes, was identified; they were significantly up-regulated during the developmental shift and inversely correlated with many genes suppressed during the mature growth phase. Among them, plant-specific NAM/ATAF/CUC (NAC) transcription factors represent an interesting gene family due to their key role in the biological processes in plant development and stress responses (Jensen et al., 2014). Five NAC genes were selected for functional characterization as key factor candidates of the major transcriptome reprogramming during grapevine development. VvNAC11, VvNAC13, VvNAC33 and VvNAC60 were identified as ‘switch’ genes in the above-mentioned analysis whereas VvNAC03 was selected because it is a close homologue of tomato NOR (non-ripening), known for its crucial role in tomato fruit ripening regulation (Giovannoni, 2004; Giovannoni et al., 1995). Firstly, the five transcription factors were transiently over-expressed in Vitis vinifera to get an overview of their primary effects on native species. Secondly, we obtained grapevine plants that were stably transformed with VvNAC33 and VvNAC60 and subjected to molecular/phenotypic characterizations. VvNAC33 seemed to be involved in negative regulation of photosynthesis since over-expressing leaves revealed a chlorophyll breakdown, while VvNAC60 affected regular plant development, showing a slight growth and earlier stem lignification in comparison to a same-age plant control. These results reflected typical behaviors of plants undergoing ripening and senescence, thus supporting our working hypothesis proposing a crucial role of NACs in the transition from vegetative to mature development in grapevine. In order to identify downstream targets of the NAC transcription factors analyzed in this work, we performed microarray analysis on leaves of transient and stable ectopic expressing plants. We noted that both over-expressions affected a wide range of cellular processes and among the most represented functional categories we found transport, secondary metabolism and transcription factor activity. The identification of VvMYBA1, a known grapevine regulator of the anthocyanin biosynthetic pathway (Kobayashi et al., 2002), as VvNAC60 target suggests a VvNAC60 role in processes like anthocyanin biosynthesis featured by grape berries at the onset of ripening. Another approach used to clarify NACs roles was to check the ability of VvNACs to fulfil the tomato NOR function. Preliminary results revealed that VvNAC03 and VvNAC60 could partially complement the nor mutation in tomato, establishing a partial ripening phenotype in fruits. Taken together, these findings suggest the ability of the selected VvNACs to affect the expression of genes involved in the regulatory network that controls the developmental shift to a mature phase in grapevine. This work has shed some light on the roles of these NACs in grapevine development, but further analysis must be conducted to fully elucidate the molecular machinery in this complex regulation system.

Grapevine (Vitis vinifera L.) is one of the most important fruit crops as it is widely cultivated, and the winemaking industry has a huge worldwide economic relevance. The global warming has affected viticulture altering the maturation processes; in particular, the anticipation of the onset of the berries ripening (veraison) has changed the physiological characteristics of grapes and, consequently, has negatively influenced the wine quality. In this contest, uncovering the molecular mechanisms of the ripening could provide the key in maintaining high quality grapes and wine. For all these reasons, the identification and characterization of master regulators controlling the transition from vegetative-to-mature growth are the challenging but fundamental tasks of this research project. At first, to provide insight into the transcriptional programs controlling the development of grapevine, a global gene expression atlas was generated (Fasoli et al., 2012). Combining this dataset with a berry-specific one (Massonnet et al., 2017), an integrated network analysis was performed (Palumbo et al., 2014) and a new category of genes (switch genes), which are significantly up regulated during the developmental shift and inversely correlated with many genes suppressed during the mature growth phase, was identified. Moreover, many transcription factors are present among them, strongly indicating that they could represent master regulators of the developmental phase transition; between them, the plant-specific NAC (NAM/ATAF/CUC) transcription factors represent an interesting family due to their key role in plant development processes and stress responses (Jensen et al., 2014). Fourteen VviNACs genes were selected for functional characterization as key candidates of the major transcriptome reprogramming during grapevine development: VviNAC01, VviNAC03 (D’Incà, 2017), VviNAC08, VviNAC11 (D’Incà, 2017), VviNAC13 (D’Incà, 2017), VviNAC15, VviNAC17, VviNAC18, VviNAC26, VviNAC33 (D’Incà, 2017), VviNAC38, VviNAC39, VviNAC60 (D’Incà, 2017) and VviNAC61. Different approaches have been carried out to investigate the function of VviNACs gene: the transient over expressions, DAP-seq (DNA Affinity Purification and sequencing, Bartlett et al., 2017) and, only for VviNAC60, the ChIP-seq (Chromatin Immunoprecipitation sequencing, Kaufmann et al., 2010). All of them are powerful techniques used to identify possible targets of the selected transcription factor regulations. VviNAC01 showed its important role in the ethylene pathways; VviNAC03 did not reveal a well-defined identity but seems to be plant growth related; VviNAC08 seems to have a possible role in the gibberellin-related and circadian mechanisms; VviNAC11 seems to be related to the control of the auxin pathways and the chlorophylls degradation; VviNAC13 revealed a probable action in the lignin and phenylpropanoid metabolic processes; VviNAC15, regulating many other TFs, highlighted its role in the regulation mechanisms orchestration; VviNAC17 appeared to be a regulator of the jasmonic acid-induced gene expression; VviNAC18 analyses reported its role in the chlorophyll degradation; VviNAC26 presented the regulation of many genes related to the sugars biosynthesis and the anthocyanin synthesis; VviNAC33 terminates the photosynthetic activity and organ vegetative growth; no data are available for VviNAC38; VviNAC39 resulted to up regulate the transport of sugar and lipids and the ubiquitin-conjugating; VviNAC60 revealed a lot of hormones related up regulated direct target genes and many transcription factors, highlighting again the important and major role of this transcription factor in the grapevine maturation processes; VviNAC61 revealed a predominant role in the regulation of the aromatic compounds biosynthesis. Concerning VviNAC60, ChIP-seq data were also obtained and one interesting gene, the SRG1- SENESCENCE-RELATED GENE 1 OXIDOREDUCTASE (VIT_10s0003g02400), was found. In order to define a regulation and co-regulation network between VviNACs, some candidate targets genes taken from the different DAP-seq datasets (VviNAC01, VviNAC05, VviNAC08, VviNAC34, VviNAC37 and VviNAC61) were tested by Dual Luciferase Reporter Assays to see by which of the selected TFs were actually regulated. The obtained results showed that VviNAC01 directly repressed VviNAC05 expression, whereas activated the VviNAC08 one; moreover, VviNAC01 was validated as repressor of its own transcription. VviNAC03 resulted a repressor of VviNAC05. VviNAC11 directly up regulated VviNAC34, VviNAC37 and VviNAC61. VviNAC13 resulted to regulate VviNAC34 and VviNAC37 expression. VviNAC15 activated VviNAC34. VviNAC17 acted as a repressor of VviNAC05, VviNAC08 and VviNAC61 expression. VviNAC18 resulted a direct activator of VviNAC05. VviNAC26 positively regulated the expression of VviNAC05 and directly down regulated the VviNAC08, VviNAC34 and VviNAC61 expression. VviNAC33 was found as a direct activator of VviNAC05, VviNAC08, VviNAC34, VviNAC37 and VviNAC61 expression. VviNAC60 showed to induced VviNAC05, VviNAC34 and VviNAC61 expression. This PhD thesis lights up the possible roles of some VviNACs in the grapevine development and presents a preliminary regulatory network between this family members; further analysis must be conducted to completely elucidate this complex regulation system.

碩士
國立臺灣大學
植物科學研究所
107
The reciprocal regulation across different transcription factors (TFs) families by intron-retained splice variants were first reported in vascular development associated VNS family members, one of the subfamily in plant-specific NAC family. Two of the VNS members, PtrSND1-A2 and PtrVND6-C1, in Populus trichocarpa undergo last intron retention (IR) and the transcripts yielded C-terminal truncated proteins with incomplete NAC domains and no activation domain. Such truncated proteins were found as dominant-negatives, suppressing the full-size proteins translated from the normally spliced transcript. To understand whether the intron retentions are conservative events throughout the VNS family in plant kingdom, we developed a Python package, MoBIReF (Motif based intron retention finder), for the transcriptomic analysis of Illumina RNA-seq to detects the conserved IR events. Total 15 species from bryophyte, pterophyte, and angiosperm were used for MoBIReF. The conserved IR events were discovered in 13 vascular species from pterophyte and angiosperm, but were not observed in the non-vascular species, the bryophyte. The synchronization between the emergence of VNSIR and vascular plants suggested that the conserved IR events may participate in the development of the vascular tissue. We further investigate such conserved IR events throughout the whole NAC family. A total of 96 NAC genes were found to possess the conserved IR events across 14 species, including the early land plant, Physcomitrella patens (moss). In this study, we demonstrated the conserved IR events are widespread in VNS and even NAC families and preserved in land plant evolution. The IR events may be general mechanism to maintain the homeostasis of VNS and NAC families for vascular development and other plant-growth related mechanisms.

碩士
中國醫藥大學
營養學系碩士班
97
Oral cancer is the fifth most frequent cancer worldwide while its mortality rate is the highest among all cancers. In Taiwan, the incidence of oral cancer increases yearly, especially in men. Despite the therapy stratagem has been advanced dramatically, the prognosis of survival rate is still relatively low. Immunohistochemical examination showed over-expressed EGFR staining in 55% to 100 % of head and neck squamous cell carcinomas (HNSCC). EGFR overexpression is associated with poor prognosis, tumor differentiation, and consequently poor survival. Hence, finding compounds that can efficiently inhibit the EGFR signaling pathway has become a promising strategy in oral cancer therapy. NAC (N-acetylcysteine), known as an anti-inflammatory factor, has anti-EGFR function, but the mechanism is still unclear. Therefore, we hypothesized that NAC inhibits EGFR signaling pathway through induction of the transcription factor HBP1 in oral cancer. We chose HSC-3 oral cancer cell line as the study model due to the fact that HSC-3 has abundant EGFR expression but relatively low HBP1 expression. As results, we showed that NAC induces HSC-3 cells arrest in G1 accompanied with decreased cyclin D1 and EGFR activation. More importantly, HBP1 expression was induced in HSC-3 cells by NAC treatment in a dose-dependent manner. Moreover, NAC also inhibited the gene expression of p47phox, one of the NADPH oxidase subunit, and thereby suppressed ROS generation in oral cancer. Further, to test if NAC inhibits EGFR activation through induction of HBP1, we employed HBP1 shRNA to knockdown endogenous HBP1 gene in HSC-3 cells. Results indicated that HBP1 reduction leads to higher EGF-stimulated EGFR and downstream Akt activation than the control one. The most importantly, HBP1 knockdown abolished NAC-inhibited EGFR activation. Thus, our results may support the transcription factor HBP1 as a future biomarker and therapeutic target in oral cancer stratagem.